Proteins, their production and use

ABSTRACT

This invention relates to a novel metalloprotease having a proteolytic activity, its partial peptide or a salt either of them, a DNA coding for the protein, a recombinant vector comprising the DNA, a transformant carrying the recombinant vector, a process for producing the protein, a pharmaceutical composition comprising the DNA, an antibody against the protein, a method for screening for a compound which activates or inhibits a proteolytic activity of the protein, a kit for screening for the compound, and a compound which activates or inhibits a proteolytic activity of the protein which is identified by the screening method or the kit. The DNA coding for the protein of the present invention can be used as a therapeutic and prophylactic composition for a variety of diseases including diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis and herniated disk. Furthermore, the protein of the present invention is useful as a screening reagent for any compounds which activate or inhibit the function of the protein of the present invention. In addition, the antibody against the protein of the present invention specifically recognizes the protein of the present invention and can be used in the quantitative determination of the protein of the present invention in a test fluid.

This application is a divisional of U.S. Ser. No. 09/171,545 filed Jul. 26, 1999 which claims the benefit of PCT/JP97/01433 filed Apr. 24, 1997, now U.S. Pat. No. 6,566,116 which in turn claims priority to Japanese application, number 8-104902, filed in Japan on Apr. 25, 1996.

TECHNICAL FIELD

The present invention relates to a novel matrix metalloprotease and a DNA coding for the metalloprotease.

BACKGROUND ART

The extracellular matrix, which is a cell-supporting tissue composed mainly of collagens and proteoglycans, is profoundly involved in such events as cell development, inflammation, and tissue repair. The enzymes known to be associated with the degradation of extracellular matrix are (1) cathepsin D, etc. which belongs to the aspartic proteaseas, (2) cathepsin B, H, L, etc. which belong to the cysteine proteases, (3) plasmin, kallikrein, neutrophil elastase, tryptase, chymase, cathepsin G, etc. which belong to the serine proteases, and (4) metalloproteases are known. Also called matrix metalloproteases, these metalloproteases are known to be playing central roles in the degradation of extracellular matrix.

So far, in humans, 13 kinds of matrix metalloproteases such as collagenases, gelatinases stromelysins, and membrane-type matrix metalloproteases have been cloned and their nucleotide sequences and amino acid sequences have been reported (T. Takino et al., Journal of Biological Chemistry, 270, 23013, 1995; J. M. P. Freije et al., Journal of Biological Chemistry, 269, 16766, 1994; H. Wills et al., European Journal of Biochemistry, 231, 602, 1995). All of these enzymes are zinc-dependent metalloproteases, in which the amino acid sequence of the zinc-binding domain: His-Glu-X-Gly-His-Ser-Leu-Gly-Leu-X-His-Ser is well conserved, and their activities are inhibited by o-phenanthroline. Each of these enzymes is secreted in the latent form which is inactive with a propeptide at the N-terminus of the active enzyme. A conserved domain consisting in the amino acid sequence of Met-Arg-Lys-Pro-Arg-Cys-Gly-Val-Pro-Asp is located near the C-terminal region of the propeptide. This domain is called “cysteine switch”, and it controls a protease activity by coordinating the zinc atom at active center with cysteine in the domain. While the latent enzymes are activated upon cleavage of the propeptide, three kinds of inhibitor proteins, named TIMP, have been reported and known to performing strict control of activity. It is also known that, in vitro, the latent enzymes are activated by treatment with trypsin or aminophenyl-mercuric acetate.

Matrix metalloproteases are not only involved in the degradation of the extracellular matrix such as collagens, gelatins which are denatured collagens, proteoglycans, fibronectins, laminins, elastins, etc. but also are in charge of activation of other matrix metalloproteases and inactivation of protease inhibitors such as α1-protease inhibitor. Furthermore, it is known that these metalloproteases are associated with solubilization of membrane proteins and cell surface proteins such as TNF, Fas ligand, IL-6 receptor, TNF-receptor, etc. and, as a result, modulate the death, differentiation, proliferation inhibition, proliferation and gene expression of cells.

It is known that physiologically matrix metallo protease activities are elevated in ovulation, development and differentiation, osteogenesis, atretic uterus, vascularization, and other events. In morbid states, those metalloprotease activities are elevated in rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), peridontitis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature labor, and atherosclerosis, among other conditions. Conversely, it is known that the enzyme activities are suppressed in fibroid lung, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, etc. Recently the fourth membrane-type matrix metalloprotease has been cloned (X. S. Puente et al., Cancer Research, 56, 944, 1996), suggesting the likelihood that there exist still other novel matrix metalloproteases.

Any novel matrix metalloproteases of human origin make it possible to develop new drugs which inhibit or stimulate the activity of the metalloprotease and are useful for the prevention and treatment of various matrix metalloprotease-associated morbidities, such as rheumatoid arthritis, and osteoarthritis. Therefore, in the technological area to which the present invention pertains, there has been a standing need for isolating novel human matrix metalloproteases and developing a method for high production of the proteins.

The inventors of the present invention have made extensive research for solving the above problems and succeeded in cloning cDNAs each having a novel nucleotide sequence from human liver-derived and rat liver-derived cDNA libraries. They have found that the proteins encoded by these cDNAs are matrix metalloproteases. The present inventors have made further investigations based on these findings, and accomplished the present invention.

DISCLOSURE OF INVENTION

The present invention provides:

-   (1) A protein comprising an amino acid sequence represented by SEQ     ID NO:1 or a substantially equivalent thereto, or a salt thereof, -   (2) The protein according to claim 1, which comprises an amino acid     sequence represented by SEQ ID NO:2, -   (3) The protein according to (1), which is a metalloprotease, -   (4) A partial peptide of the protein according to (1), or a salt     thereof, which shows the activity of the protein according to (1), -   (5) An isolated DNA which contains a DNA comprising a nucleotide     sequence coding for a protein according to (1), -   (6) The DNA according to (5), which comprises a nucleotide sequence     represented by SEQ ID NO:4, -   (7) The DNA according to (5), which comprises a nucleotide sequence     represented by SEQ ID NO:8, -   (8) A recombinant vector comprising the DNA according to (5), -   (9) A transformant carrying the recombinant vector according to (8), -   (10) A process for producing a protein or a salt thereof according     to (1), which comprises culturing a transformant according to (9)     under conditions suitable to express the protein, -   (11) A pharmaceutical composition which comprises the protein     according to (1) or the partial peptide according to (4), -   (12) The pharmaceutical composition according to (11) which is a     therapeutic or prophylactic composition for diabetic nephropathy,     glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis,     hepatocirrhosis, osteopetrosis or herniated disk, -   (13) A pharmaceutical composition which comprises the DNA according     to (5), -   (14) The pharmaceutical composition according to (13) which is a     therapeutic or prophylactic composition for diabetic nephropathy,     glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis,     hepatocirrhosis, osteopetrosis or herniated disk, -   (15) An antibody against the protein according to (1) or the partial     peptide according to (4), -   (16) A method for screening for a compound which activates or     inhibits a proteolytic activity of the protein according to (1) or     the partial peptide according to (4), which comprises measuring and     comparing a proteolytic activity of the protein according to (1) or     the partial peptide according to (4), in case of (i) a substrate is     contacted with the protein according to (1) or the partial peptide     according to (4) and (ii) a substrate and a test compound are     contacted with the protein according to (1) or the partial peptide     according to (4), -   (17) A kit for screening for a compound which activates or inhibits     a proteolytic activity of the protein according to (1) or the     partial peptide according to (4), which comprises the protein     according to (1) or the partial peptide according to (4), -   (18) A compound which activates or inhibits a proteolytic activity     of the protein according to (1) or the partial peptide according to     (4), which is identified by the screening method according to (16)     or the kit according to (17), -   (19) A pharmaceutical composition which comprises the compound which     inhibits a proteolytic activity of the protein according to (1) or     the partial peptide according to (4), which is identified by the     screening method according to (16) or the kit according to (17), -   (20) A method for treating or preventing diabetic nephropathy,     glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis,     hepatocirrhosis, osteopetrosis or herniated disk in a mammal, which     comprises administering an effective amount of the protein according     to (1) or the partial peptide according to (4), -   (21) Use of the protein according to (1) or the partial peptide     according to (4) for production of a therapeutic or prophylactic     composition for diabetic nephropathy, glomerulonephritis, pulmonary     fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis or     herniated disk, -   (22) A method for treating or preventing diabetic nephropathy,     glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis,     hepatocirrhosis, osteopetrosis or herniated disk in a mammal, which     comprises administering an effective amount of the DNA according     to (5) to the mammal, and -   (23) Use of the DNA according to (5) for production of a therapeutic     or prophylactic composition for diabetic nephropathy,     glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis,     hepatocirrhosis, osteopetrosis or herniated disk.

Moreover, the present invention provides:

-   (24) The partial peptide according to (4), which comprises an amino     acid sequence represented by any one of SEQ ID NO:3 to SEQ ID NO:6     or a substantial equivalent thereto, -   (25) An isolated DNA which hybridizes under highstringent condition     to a DNA comprising a nucleotide sequence represented by SEQ ID NO:7     or SEQ ID NO:8, -   (26) A recombinant vector comprising the DNA according to (25), -   (27) A transformant carrying the recombinant vector according to     (26), -   (28) A process for producing a protein which is encoded by the DNA     according to (25) or a salt thereof comprising culturing a     transformant according to (27) under conditions suitable to express     the protein, -   (29) A protein produced by the process according to (28), -   (30) The pharmaceutical composition according to (19) which is a     therapeutic or prophylactic composition for wound, rheumatoid     arthritis, osteoarthritis, cancer (metasrasis and invasion),     periontitis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm,     otosclerosis, epidermolysis bullosa, premature delivery,     atherosclerosis, septemia, multiple (disseminated) sclerosis,     cachexia, hypercalcemia, leulemia, lymphoma, diabetes, systemic     lupus erythematosus, asthma, allergic rhinitis, atopic dermatitis,     trauma, burn, acute pancreatitis, ischemia-reperfusion syndrome,     myocardial infarction, congrestive heart failure, organ     transplantation or graft-vs.-host disease (GVHD), -   (31) A pharmaceutical composition which comprises the compound which     activates a proteolytic activity of the protein according to (1) or     the partial peptide according to (4), or a salt thereof, which is     identified by the screening method according to (16) or the kit     according to (17), -   (32) The pharmaceutical composition according to (31) which is a     therapeutic or prophylactic composition for diabetic nephropathy,     glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis,     hepatocirrhosis, osteopetrosis or herniated disk, -   (33) A method of quantitative determination of the protein according     to (1) or the partial peptide according to (4) in a test liquid     sample, which comprises -   (a) competitively reacting the test liquid sample and a labeled     protein according to (1) or partial peptide according to (4) with     the antibody according to (15), and -   (b) measuring the ratio of the labeled protein according to (1) or     partial peptide according to (4) which binds to the antibody, and -   (34) A method of quantitative determination of the protein according     to (1) or the partial peptide according to (4) in a test liquid     sample, which comprises -   (a) reacting the test liquid sample with the antibody according     to (15) immobilized on an insoluble carrier and a labeled antibody     according to (15) simultaneously or continuously, and -   (b) measuring the activity of the labeling agent on the insoluble     carrier.

The protein comprising the amino acid sequence represented by SEQ ID NO:1 (FIG. 1) or a substantial equivalent thereto of the present invention (hereinafter referred to as the protein of the present invention) may be (1) a protein derived from cells of human and other warm-blooded animals (e.g. guinea pig, rat, mouse, chicken, rabbit, swine, sheep, bovine, monkey, etc.) such as liver cell, splenocytes, nerve cell, glia cell, B cell, bone marrow cell, mesangial cell, Langerhans' cell, epidermic cell, epithelial cell, endothelial cell, fibroblast, fibrocyte, myocyte, fat cell, immune cell (e.g. macrophage, T cell, B cell, natural killer cell, mast cell, neutrophil, basophil, eosinophil, monocyte), megakaryocyte, synovial cell, chondrocyte, bone cell, osteoblast, osteoclast, mammary gland cell, hepatocyte, interstitial cell, etc., the corresponding precursor cells, stem cells, cancer cells, etc., or any tissues where such cells are present, such as brain or any of its regions (e.g. olfactory bulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata, cerebellum, etc.), spinal cord, hypophysis, stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g. large intestine and small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc., (2) a protein derived from cultured human cell lines (e.g. MEL, M1, CTLL-2, HT-2, WEHI-3, HL-60, JOSK-1, K562, ML-1, MOLT-3, MOLT-4, MOLT-10, CCRF-CEM, TALL-1, Jurkat, CCRT-HSB-2, KE-37, SKW-3, HUT-78, HUT-102, H9, U937, THP-1, HEL, JK-1, CMK, KO-812, MEG-01, etc.), or (3) a synthetic protein.

Examples of the substantially equivalent amino acid sequence to the amino acid sequence represented by SEQ ID NO:1 are an amino acid sequence of not less than about 50%, preferably not less than about 60%, more preferably not less than about 70%, further more preferably not less than about 80%, for still better result, not less than about 90%, best example, not less than about 95% identity to the amino acid sequence represented by SEQ ID NO:1 and so on. More preferable examples are an amino acid sequence of not less than about 50%, preferably not less than about 60%, more preferably not less than about 70%, further more preferably not less than about 80%, for still better result, not less than about 90%, best example, not less than about 95% identity to the amino acid sequence represented by SEQ ID NO:1, which comprises at least an amino acid sequence represented by SEQ ID NO:3 (FIG. 2) or SEQ ID NO:4 (FIG. 3), and so on.

Examples of the substantially equivalent amino acid sequence to the amino acid sequence represented by SEQ ID NO:1 are an amino acid sequence represented by SEQ ID NO:2, and so on.

Examples of the protein comprising a substantially equivalent to the amino acid sequence represented by SEQ ID NO:1 are a protein which comprises a substantially equivalent amino acid sequence to the amino acid sequence represented by SEQ ID NO:1 and has a substantially equivalent activity to the protein comprising the amino acid sequence represented by SEQ ID NO:1, and so on.

More preferable examples of the protein are a protein comprising an amino acid sequence represented by SEQ ID NO:2, and so on.

Examples of the substantially equivalent activity are a proteolytic activity (e.g. activity of proteases such as proteinases, peptidases, etc.) and so on. The term “substantially equivalent” means that the nature of these activities are physiologically chemically or phramacologically equivalent. Therefore, it is preferred that the strength of activities such as a proteolytic activity is equivalent (e.g. about 0.01 to 100 times, preferably about 0.5 to 20 times, more preferably about 0.5 to 2 times), and it is allowable that even differences among grades such as the strength of these activities and molecular weight of the protein are present.

Activities such as a proteolytic activity may be measured by per se known methods. For example, the proteolytic activity may be measured by the method for screening as mentioned below.

The proteins of the present invention include muteins such as proteins comprising (1) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) are deleted from the amino acid sequence represented by SEQ ID NO:1, (2) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferable 1 to 10, more preferable a few (1 to 5) amino acid residues) are added to the amino acid sequence represented by SEQ ID NO:1, (3) an amino acid sequence wherein 1 or more than acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) in the amino acid sequence represented by SEQ ID NO:1 are substituted with 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues), or (4) combinations thereof.

When the amino acid sequence of the proteins are deleted or substituted as mentioned above, examples of the positions of deletion or substitution are, for example, (1) other than 98th to 508th amino acid sequence of the amino acid sequence represented by SEQ ID NO:1 (other than an amino acid sequence represented by SEQ ID NO:3), more preferably, other than 93rd to 508th amino acid sequence of the amino acid sequence represented by SEQ ID NO:1 (an amino acid sequence represented by SEQ ID NO:4), or (2) other than 99th to 517th amino acid sequence of the amino acid sequence represented by SEQ ID NO:2 (other than an amino acid sequence represented by SEQ ID NO:5), more preferably, other than 94th to 517th amino acid sequence of the amino acid sequence represented by SEQ ID NO:2 (other than an amino acid sequence represented by SEQ ID NO:6). Other preferable examples of the positions of deletion or substitution are other than a common sequence of the amino acid sequence represented by SEQ ID NO:1 and the amino acid sequence represented by SEQ ID NO:2, and more preferable examples are, for example, other than 212nd to 225th amino acid sequence of the amino acid sequence represented by SEQ ID NO:1 (that is, other than 213rd to 226th amino acid sequence of the amino acid sequence represented by SEQ ID NO:2).

Throughout this specification, proteins are represented in accordance with the conventions for description of peptides, that is the N-terminus (amino terminus) at left and the C-terminus (carboxyl terminus) at right. The protein of the present invention including the protein containing the amino acid sequence of SEQ ID NO:1 is usually in the carboxyl (—COOH) or carboxylate (—COO⁻) form at the C-terminus but may be in the amide (—CONH₂) or ester (—COOR) form.

R in the ester residue includes a C₁₋₆ alkyl group (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, etc., a C₃₋₈ cycloalkyl group (e.g. cyclopentyl, cyclohexyl, etc.), a C₆₋₁₂ aryl group (e.g. phenyl, α-naphthyl, etc.), a C₇₋₁₄ aralkyl group such as a phenyl-C₁₋₂ alkyl group (e.g. benzyl, phenethyl, etc.) and α-naphthyl-C₁₋₂ alkyl, (e.g. α-naphthylmethyl, etc.), as well as pivaloyloxymethyl which is universally used for the production of esters for oral administration.

When the protein of the present invention has a carboxyl (or carboxylate) function in any position other than the C-terminus, the corresponding carboxamide or ester form is also included in the scope of the invention. The ester mentioned just above may be any of the esters mentioned for the C-terminal carboxyl function.

Furthermore, the protein of the present invention includes (1) the protein in which the N-terminal Met has been protected with a protective group (e.g. C₁₋₆ acyl such as formyl or C₁₋₅ alkyl-carbonyl such as acetyl, etc.), (2) the protein in which the N-terminal side of Glu has been cleaved in vivo to form pyroglutamic acid, (3) the protein in which the side chain of any relevant constituent amino acid (e.g. OH, COOH, NH₂, SH) has been protected by any protective group (e.g. C₁₋₆ acyl group such as formyl or C₁₋₅ alkyl-carbonyl group such as acetyl, etc.), and (4) the complex protein such as glycoproteins available upon attachment of sugar chains.

Preferable Examples of the proteins of the present invention are human metalloproteases such as a human liver-derived metalloprotease comprising an amino acid sequence represented by SEQ ID NO:1 (FIG. 1), rat metalloproteases such as a rat liver-derived metalloprotease comprising an amino acid sequence represented by SEQ ID NO:2 (FIG. 6).

Examples of the partial peptide of the present invention are any partial peptides of the protein of the present invention as mentioned above which have a proteolytic activity. For example, the partial peptides include peptides comprising at least not less than about 20, preferably not less than about 50, more preferably not less than about 70, for still better result, not less than about 100, best result, not less than 200 amino acid residues of the amino acid sequence of the proteins of the present invention.

Preferable examples of the partial peptide of the present invention are (1) a peptide which comprises an amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:4, or a substantially equivalent thereto and has a substantially equivalent activity to the protein comprising the amino acid sequence represented by SEQ ID NO:1, (2) a peptide which comprises an amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:6, or a substantially equivalent thereto and has a substantially equivalent activity to the protein comprising the amino acid sequence represented by SEQ ID NO:2.

Examples of the substantially equivalent amino acid sequence to the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:4 are an amino acid sequence of not less than about 50%, preferably not less than about 60%, more preferably not less than about 70%, further more preferably not less than about 80%, for still better result, not less than about 90%, best example, not less than about 95% identity to the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:4.

Examples of the substantially equivalent amino acid sequence to the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:6 are an amino acid sequence of not less than about 50%, preferably not less than about 60%, more preferably not less than about 70%, further more preferably not less than about 80%, for still better result, not less than about 90%, best example, not less than about 95% identity to the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:6.

The amino acid sequence represented by SEQ ID NO:3 shows an amino acid sequence from ⁹⁸Tyr to ⁵⁰⁸Tyr of the amino acid sequence represented by SEQ ID NO:1, and the amino acid sequence represented by SEQ ID NO:4 shows an amino acid sequence from ⁹³Gln to ⁵⁰⁸Tyr of the amino acid sequence represented by SEQ ID NO:1. The both amino acid sequences show amino acid sequences of catalytic domain of the protein of the present invention, and show amino acid sequence of the mature protein of the protein of the present invention comprising the amino acid sequence represented by SEQ ID NO:1.

The amino acid sequence represented by SEQ ID NO:5 shows an amino acid sequence from ⁹⁹Tyr to ⁵¹⁷Tyr of the amino acid sequence represented by SEQ ID NO:12, and the amino acid sequence represented by SEQ ID NO:6 shows an amino acid sequence from ⁹⁴Gln to ⁵¹⁷Tyr of the amino acid sequence represented by SEQ ID NO:12. The both amino acid sequences show amino acid sequences of catalytic domain of the protein of the present invention, and show amino acid sequence of the mature protein of the protein of the present invention comprising the amino acid sequence represented by SEQ ID NO:2.

The protein of the present invention is expressed as the protein comprising the amino acid sequence represented by SEQ ID NO:1, and is cleaved in vivo at the position of 1st to 97th or 1st to 92nd of the amino acid sequence represented by SEQ ID NO:1. And the peptide comprising 98th to 508th amino acid sequence (SEQ ID NO:3, FIG. 2) or 93rd to 508th amino acid sequence (SEQ ID NO:4, FIG. 3) of the amino acid sequence represented by SEQ ID NO:1, etc. show a proteolytic activity, etc. as a mature protein or active protein.

The protein of the present invention is expressed as the protein comprising the amino acid sequence represented by SEQ ID NO:2, and is cleaved in vivo at the position of 1st to 98th or 1st to 93rd of the amino acid sequence represented by SEQ ID NO:2. And the peptide comprising 98th to 508th amino acid sequence (SEQ ID NO:5) or 94th to 517th amino acid sequence (SEQ ID NO:6) of the amino acid sequence represented by SEQ ID NO:2, etc. show a proteolytic activity, etc. as a mature protein or active protein.

The term “substantially equivalent activity” has the same meaning as defined above. The “substantially equivalent activity” can be measured by the same method as mentioned above.

The partial peptide of the present invention may include peptides such as peptide comprising (1) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) are deleted from the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:4, (2) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferable 1 to 10, more preferable a few (1 to 5) amino acid residues) are added to the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:4, (3) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) in the amino acid sequence represented by SEQ ID NO:3 or SEQ ID NO:4 are substituted with 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues), or (4) combinations thereof.

Other partial peptide of the present invention may include peptides such as peptide comprising (1) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) are deleted from the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:6, (2) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferable 1 to 10, more preferable a few (1 to 5) amino acid residues) are added to the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:6, (3) an amino acid sequence wherein 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues) in the amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:6 are substituted with 1 or more amino acid residues (for example 1 to 30, preferably 1 to 10, more preferably a few (1 to 5) amino acid residues), or (4) combinations thereof.

The peptide of the present invention is usually in the carboxyl (—COOH) or carboxylate (—COO⁻ form at the C-terminus, but may instead be in the amide (—CONH₂) or ester (—COOR) form as same as the protein of the present invention as mentioned above.

Furthermore, the partial peptide of the present invention includes (1) the peptide in which the N-terminal Met has been protected with a protective group, (2) the peptide in which the N-terminal side of Glu has been cleaved in vivo to form pyroglutamic acid, (3) the peptide in which the side chain or any relevant constituent amino acid has been protected by any protective group, and (4) the complex peptide such as glycoproteins available upon attachment of sugar chains as same as the protein of the present invention as mentioned above.

The specific examples of the partial peptide of the present invention are a peptide comprising an amino acid sequence represented by SEQ ID NO:3 (FIG. 2) or SEQ ID NO:4 (FIG. 3), a peptide comprising an amino acid sequence represented by SEQ ID NO:5 or SEQ ID NO:6, and so on.

The salt of the protein or the partial peptide of the present invention includes salts with physiologically acceptable bases, e.g. alkali metals or acids such as organic or inorganic acids, and is preferably a physiologically acceptable acid addition salt. Examples of such salts are salts thereof with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid, etc.) and salts thereof with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid or benzenesulfonic acid, etc.)

The protein or a salt thereof of the present invention can be produced from the tissues or cells of human or other warm-blooded animals by per se known purification technology or, as described hereinafter, by culturing a transformant carrying a DNA encoding the protein. It can also be produced in accordance with the procedures for peptide synthesis which are described hereinafter.

When the protein of the present invention is produced from the tissues or cells of human or other warm-blooded animals, the tissues or cells of human or other warm-blood animals are homogenized and the protein of the present invention is extracted by an acid, etc. The protein can be purified and isolated from the extracted solution by a combination of chromatography such as reverse phase chromatography, ion exchange chromatography and so on.

For the synthesis of the protein of the present invention, a partial peptide thereof or their salts, or their amides form, any of commercial resins available for protein synthesis can be employed. Among such resins are chloromethyl resin, hydroxymethyl resin, benzhydrylamino resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamino resin, PAM resin, 4-hydroxymethyl-methylphenylacetamidomethyl resin, polyacrylamide resin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin, and 4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin. Using such a resin, amino acids which may be beforehand protected at side-chain functional groups in a suitable manner can be serially condensed with the a-amino group in the order corresponding to the amino acid sequence of the objective protein by various condensation techniques which are per se known. After completion of the final condensation reaction, the protein is separated from the resin and the protective groups are removed. Then, in highly diluted solution, the intramolecular disulfide-forming reaction is carried out to provide the objective proteins or amides thereof.

Referring to the above condensation of protected amino acids, various activating agents known to be useful for protein synthesis can be utilized, and carbodiimide reagents are especially preferred. The carbodiimide reagents include are DCC, N,N′-diisopropylcarbodiimide, and N-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide and so on. For activation by these reagents, the protected amino acid and a racemization inhibitor (e.g. HOBt, HOOBt, etc.) can be directly added to the resin, or the protected amino acid can be activated beforehand in the form of symmetric acid anhydride, HOBt ester or HOOBt ester and, then, added to the resin.

The solvent used for the above-mentioned activation of protected amino acids or the conjugation thereof to the resin can be optionally selected from among the solvents known to be useful for protein condensation reactions. Examples of the solvent are acid amides (e.g. N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc.), halogenated hydrocarbons (e.g. methylene chloride, chloroform, etc.), alcohols (e.g. trifluoroethanol, sulfoxides (e.g. dimethyl sulfoxide, etc.), ethers is (e.g. pyridine, dioxane, tetrahydrofuran, etc.), nitrites (e.g. acetonitrile, propionitrile, etc.), esters (e.g. methyl acetate, ethyl acetate, etc.), and suitable mixtures of these solvents. The reaction temperature can be selected from the range known to be useful for protein-forming reactions, usually the range of about −20° C. to about 50° C. The activated amino acid derivative is generally used in a 1.5 to 4-fold excess. When the condensation is found insufficient by ninhydrin assay, the reaction can be repeated to make the sufficient condensation thorough. When sufficient condensation can not be achieved by repeated reaction, an unreacted amino acid can be acetylated by using acetic anhydride or acetylimidazole so as not to effect a subsequent reaction.

The protective groups for protecting the amino group of the starting compound include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxy-benzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc, and so on.

The carboxyl group can be protected in the form of, for example, an alkyl ester (e.g. straight-chain, branched, or cyclic alkyl esters such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, and so on), an aralkyl ester (e.g. benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, benzhydryl, and so on), phenacyl ester, benzyloxycarbonylhydrazide, t-butoxycarbonylhydrazide or tritylhydrazide.

The hydroxyl group of serine can be protected in the form of an ester or an ether. The group suitable for esterification includes carboxylic acid-derived acyl groups such as a lower (C₁₋₆) alkanoyl group (e.g. acetyl, etc.), an aroyl group (e.g. benzoyl, etc.), a benzyloxycarbonyl, an ethoxycarbonyl group and so on. The group suitable for etherification includes a benzyl group, a tetrahydropyranyl group, a t-butyl group and so on.

The protective group used for protecting the phenolic hydroxyl group of tyrosine includes Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, t-butyl and so on.

The protective group for the imidazole group of histidine includes Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc and so on.

The starting compound with activated carboxyl groups includes the corresponding acid anhydride, azide, and active ester (e.g. esters with alcohols such as pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc.). The starting compound with activated amino groups includes the corresponding phosphorylamide.

The method for removal of such protective groups includes catalytic reduction in a hydrogen stream in the presence of a catalyst (e.g. Pd black or Pd-on-carbon), acid treatment with anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, treatment with a base such as diisopropylethylamine, triethylamine, piperidine, piperazine or the like, and reduction with sodium metal in liquid ammonia. The above deprotection by treatment with acid is generally conducted at a temperature of about −20° C. to 40° C. This acid treatment can be carried out advantageously in the presence of a cation acceptor such as anisole, phenol, thioanisole, m-cresol, p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, or the like. The 2,4-dinitrophenyl group used for protecting the imidazole group of histidine can be removed by treatment with thiophenol, and the formyl group used for protecting the indole group of tryptophan can be removed not only by said acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol or the like as described hereinbefore, but also by alkali treatment with diluted sodium hydroxide solution, diluted liquid ammonia, or the like.

The method for protecting any functional group that should not take part in the contemplated reaction, the protective group to be used for such protection, the method for eliminating the protective group, and the method for activating the functional group to be involved in the contemplated reaction can all be optionally selected from among the known methods and groups.

An alternative method for providing the protein in amide form typically comprises protecting the α-carboxyl group of the C-terminal amino acid in the form of an amide, extending the peptide (protein) chain to a desired length towards the N-terminus, deprotecting the N-terminal α-amino acid of the resulting peptide chain selectively to provide an N-terminal-deprotected fragment, preparing a peptide (protein) fragment with its C-terminal carboxyl group selectively deprotected, and condensing the two fragments in a solvent such as the mixed solvent as mentioned above. The condensation reaction can be carried out in the same manner as described hereinbefore. After purification of the protected protein thus obtained by condensation, all the protective groups are eliminated by the procedures described hereinbefore to provide the contemplated protein in crude form. This crude protein is purified by suitable known purification techniques and lyophilized to provide the desired protein amide.

A method for providing the protein in an ester form comprises condensing the α-carboxyl group of the C-terminal amino acid with a suitable alcohol to prepare the corresponding ester and subjecting this ester to the same procedure as described for purification of the protein amide to provide the objective protein ester.

The partial peptide of the protein of the present invention or a salt thereof can be produced by per se known procedures for peptide synthesis or by cleaving the protein with a suitable peptidase. The process for peptide synthesis may be a solid-phase synthesis and/or a liquid-phase synthesis. Namely, the objective peptide can be produced by condensing a partial peptide or amino acid capable of constituting the protein with the residual part thereof and, when the product has a protective group, the protective group is removed whereupon a desire peptide can be manufactured. The known technology for condensation and deprotection includes the procedures described in the following literature (1)-(5).

-   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis, Interscience     Publishers, New York, 1966 -   (2) Schroeder and Luebke, The Peptide, Academic Press, New York,     1965 -   (3) Nobuo Izumiya et al., Fundamentals and Experiments in Peptide     Synthesis, Maruzen, 1975 -   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment     Series 1, Protein Chemistry IV, 205, 1977 -   (5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14,     Peptide Synthesis, Hirokawa Shoten

After the reaction, the partial peptide of the present invention can be purified and isolated by a combination of conventional purification techniques such as solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization. When the partial peptide isolated as above is in a free form, it can be converted to a suitable salt by known methods or method analogous thereto. On the other hand, when it is isolated as a salt, it can be converted to a free form or to any other salt thereof by known methods or method analogous thereto.

The DNA coding for the protein of the present invention may be any DNA comprising a nucleotide sequence encoding the protein of the present invention as mentioned above. It may also be any one of genomic DNA, genomic DNA library, cDNA derived from the tissues or cells as mentioned above, cDNA library derived from the tissues or cells as mentioned above, and synthetic DNA.

The vector for constructing a library may include bacteriophage, plasmid, cosmid, and phagemid. Furthermore, using a total RNA fraction or an mRNA fraction prepared from the tissues or cells, a direct amplification can be carried out by Reverse Transcriptase Polymerase Chain (hereinafter, referred to as RT-PCR method) technique.

Examples of DNA coding for the protein of the present invention are (1) a DNA comprising a nucleotide sequence represented by SEQ ID NO:7, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:7 under a highstringent condition and codes for a protein having a substantially equivalent activity to the protein comprising the amino acid sequence represented by ID No:1, (2) a DNA comprising a nucleotide sequence represented by SEQ ID NO:8, or a DNA which comprises a nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:8 under a highstringent condition and codes for a protein having a substantially equivalent activity to the protein comprising the amino acid sequence represented by ID No:2, and so on.

Examples of the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:7 are a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:7.

Examples of the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:8 are a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:8.

The hybridization can be carried out by per se known methods such as the method described in Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989) and so on. When a commercially available library is used, the hybridization can be carried out in accordance with the instructions given in the accompanying manual, and particularly, be carried out under a highstringent condition.

Under the highstringent condition, Na⁺ concentration is at about 19 to 40 mM, preferably about 19 to 20 mM and a temperature is at about 50 to 70° C., preferably about 60 to 65° C. Particularly, the condition at about 19 mM of Na⁺ and about 65° C. is preferred.

Preferable examples of the DNA coding for the protein comprising an amino acid sequence represented by SEQ ID NO:1 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:7 (95th to 1618th nucleotide sequence of the nucleotide sequence of FIG. 4), and preferable example of the DNA coding for the protein comprising an amino acid sequence represented by SEQ ID NO:2 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:8 (90th to 1640th nucleotide sequence of the nucleotide sequence of FIG. 6).

The DNA coding for the partial peptide of the present invention may be any DNA comprising a nucleotide sequence encoding the partial peptide of the present invention as mentioned above. It may also be any one of genomic DNA, genomic DNA library, cDNA derived from the tissues or cells as mentioned above, cDNA library derived from the tissues or cells as mentioned above, and synthetic DNA.

Examples of DNA coding for the partial peptide of the present invention are (1) a DNA comprising a partial nucleotide sequence of DNA which comprises a nucleotide sequence represented by SEQ ID NO:7, or a DNA comprising a partial nucleotide sequence of DNA which comprises a nucleotide sequence hybridizing under a highstringent condition to the nucleotide sequence represented by SEQ ID NO:7 and codes for a protein having a substantially equivalent activity to the protein comprising the amino acid sequence represented by SEQ ID NO:1, (2) a DNA comprising a partial nucleotide sequence of DNA which comprises a nucleotide sequence represented by SEQ ID NO:8, or a DNA comprising a partial nucleotide sequence of DNA which comprises a nucleotide sequence hybridizing under a highstringent condition to the nucleotide sequence represented by SEQ ID NO:8 and codes for a protein having a substantially equivalent activity to the protein comprising the amino acid sequence represented by SEQ ID NO:2, and so on.

Preferable examples of DNA coding for the partial peptide of the present invention are (1) a DNA comprising a nucleotide sequence represented by SEQ ID NO:9, or a DNA which comprises a nucleotide sequence hybridizing under a highstringent condition to the nucleotide sequence represented by SEQ ID NO:9 and codes for a partial peptide having a substantially equivalent activity to the protein of the present invention, (2) a DNA comprising a nucleotide sequence represented by SEQ ID NO:10, or a DNA which comprises a nucleotide sequence hybridizing under a highstringent condition to the nucleotide sequence represented by SEQ ID NO:10 and codes for a partial peptide having a substantially equivalent activity to the protein of the present invention, (3) a DNA comprising a nucleotide sequence represented by SEQ ID NO:11, or a DNA which comprises a nucleotide sequence hybridizing under a highstringent condition to the nucleotide sequence represented by SEQ ID NO:11 and codes for a partial peptide having a substantially equivalent activity to the protein of the present invention, (4) a DNA comprising a nucleotide sequence represented by SEQ ID NO:12, or a DNA which comprises a nucleotide sequence hybridizing under a highstringent condition to the nucleotide sequence represented by SEQ ID NO:12 and codes for a partial peptide having a substantially equivalent activity to the protein of the present invention, and so on.

Examples of the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:9 or SEQ ID NO:10 are a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:9 or SEQ ID NO:10, and so on.

Examples of the nucleotide sequence hybridizing to the nucleotide sequence represented by SEQ ID NO:11 or SEQ ID NO:12 are a nucleotide sequence of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, for still better result, not less than about 95% identity to the nucleotide sequence represented by SEQ ID NO:11 or SEQ ID NO:12, and so on.

The method for hybridization and the highstringent condition have same meanings as mentioned above.

Preferable examples of the DNA coding for the partial peptide represented by SEQ ID NO:3 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:9 (386th to 1618th nucleotide sequence of the nucleotide sequence of FIG. 4) and so on. Preferable examples of the DNA coding for the protein represented by SEQ ID NO:4 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:10 (371th to 1618th nucleotide sequence of the nucleotide sequence of FIG. 4) and so on.

Preferable examples of the DNA coding for the partial peptide comprising an amino acid sequence represented by SEQ ID NO:5 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:11 (295th to 1640th nucleotide sequence of the nucleotide sequence of FIG. 6) and so on. Preferable examples of the DNA coding for the protein comprising an amino acid sequence represented by SEQ ID NO:6 are a DNA comprising the nucleotide sequence represented by SEQ ID NO:12 (280th to 1640th nucleotide sequence of the nucleotide sequence of FIG. 6) and so on.

The DNA encoding the entire protein or the partial peptide of the present invention can be cloned either by PCR amplification using synthetic DNA primers having a partial nucleotide sequence of the DNA coding for the protein or by hybridization using the DNA inserted in a suitable vector and labeled DNA fragment or synthetic DNA coding for a part or full region of the protein or the partial peptide of the present invention. The hybridization can be carried out by the method described in Molecular Cloning, 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available DNA library is used, the instructions given in the accompanying manual can be followed.

The substitution of the nucleotide sequence of the DNA can be carried out by the per se known method such as Gapped duplex method, Kunkel method and so on by using the known kits such as Mutan™-G (Takara corporation), Mutan™-K (Takara corporation) and so on.

The cloned DNA coding for the protein of the present invention can be used directly or after digestion with a restriction enzyme or after addition of a linker depending on purposes. This DNA may have ATG as the translation initiation codon at the 5′ end and TAA, TGA, or TAG as the termination codon at the 3′ end. The translation initiation and termination codons can be added by means of suitable DNA adapters.

An expression vector for the protein of the present invention can be produced by, for example, (a) cutting out an objective DNA fragment from the DNA for the protein of the present invention and (b) ligating the objective DNA fragment with the downstream side of a promoter in a suitable expression vector.

The vector may include plasmids derived from Escherichia coli, e.g., pBR322, pBR325, pUC12, pUC13, etc.; plasmids derived from Bacillus subtilis, e.g., pUB110, pTP5, pC194, etc.; plasmids derived from yeasts e.g., pSH19, pSH15, etc.; bacteriophages such as λ-phage: animal virus such as retrovirus, vaccinia virus, etc.; insect virus; and other vecters such as pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and so on.

According to the present invention, any promoter can be used as long as it is appropriate for the host cell which is used for expressing a gene. When the host is an animal cell, the promoter include SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-TK promoter, etc., and CMV promoter and SRα promoter are preferably used. When the host for the transformation is Escherichia coli, the promoter are preferably trp promoter, lac promoter, recA promoter, λP_(L) promoter, lpp promoter, T7 promoter, etc. When the host for the transformation is Bacillus, the promoter are preferably SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is a yeast, the promoter are preferably PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. When the host is an insect cell, the promoter include polyhedrin promoter, P10 promoter, etc.

The expression vectors may, if necessary, further comprise enhancers, splicing signals, polyadenylation signals, selective markers, SV40 duplicate origin (hereinafter referred to as SV40 ori). Examples of selective markers are dihydrofolic acid reductase (hereinafter referred to as dhfr gene (methotrexate (MTX) registant)), neomycin-resistant gene (hereinafter referred to as Neo, G418 resistant) and so on. Particularly, when the dhfr gene is used as a selective marker against gene-deficient chinese hamster cell lines, cells transfected by the objective gene can be selected in a thymidine-free medium.

Furthermore, an appropriate signal sequence for a host can be added to the N-terminal side of the protein. When the host is Escherichia coli, the utilizable signal sequences may include PhoA signal sequence, OmpA signal sequence, etc. When the host is Bacillus, they may include α-amylase signal sequence, subtilisin signal sequence, etc. When the host is a yeast, they may include MFα signal sequence, SUC2 signal sequence, etc. When the host is an animal cell, they may include insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc.

A transformant or transfectant is produced by using the vector thus constructed, which carries the DNA coding for the protein of the present invention.

The host may be, for example, Escherichia species, Bacillus species, yeast cells, insect cells, insects, animal cells, etc.

Examples of Escherichia species include Escherichia coli K12•DH1 (Proceedings of the National Academy of Sciences of the United State of America, Vol. 60, 160 (1968)), JM103 (Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal of Molecular Biology, Vol. 120, 517 (1978)), HB101 (Journal of molecular Biology, Vol, 41, 459 (1969)), C600 [Genetics, Vol. 39, 440 (1954)), etc.

Examples of Bacillus species are, for example, Bacillus subtilis MI114 (Gene, Vol. 24, 255 (1983)), 207-21 (Journal of Biochemistry, Vol. 95, 87 (1984)), etc.

Examples of yeast cells are, for example, Saccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D or 20B-12, Schizosachcaromyces pombe NCYC1913 or Pichia pastoris, etc.

Examples of insect cells are, for example, Spodoptera frugiperda cell (Sf cell), MG1 cell derived from center intestine of Trichoplusia ni, High Five™ cell derived from eggs of Trichoplusia ni, Mamestra brassicae-derived cell, Estigmena acrea-derived cell and so on when virus is AcNPV; and Bombyx mori N cell (BmN cell) and so on when virus is BmNPV. Examples of the Sf cell are, for example, Sf9 cell (ATCC CRL 1711), Sf21 cell [both, Vaughn J. L. et al., In Vivo, 13, 213-217(1977)] and so on.

Examples of insects include a larva of silkworm (Bombyx mori larva) (Maeda et al., Nature, 315, 592(1985)).

Examples of animal cells are, for example, monkey-derived COS-7 cell line, Vero cell line, Chinese hamster ovary cell line (hereinafter referred to as CHO cell), dhfr gene-deficient Chinese hamster cell line (hereinafter referred to as CHO(dhfr⁻) cell), mouse L cell, mouse AtT-20, mouse myeloma cell, rat GH3, human FL, 293 cell, C127 cell, BALB3T3 cell, Sp-2/O cell, etc. Among them, CHO cell, CHO(dhfr⁻) cell, 293 cell, etc. are preferred.

Depending on host cells used, transformation is done using standard techniques appropriate to such cells.

Transformation of Escherichia species can be carried out in accordance with methods as disclosed in, for example, Proceedings of the National Academy of Sciences of the United State of America, Vol. 69, 2110 (1972), and Gene, Vol. 17, 107 (1982), etc. Transformation of Bacillus species can be carried out in accordance with methods as disclosed in, for example, Molecular & General Genetics, Vol. 168, 111 (1979), etc.

Transformation of yeast cells can be carried out in accordance with methods as disclosed in, for example, Methods in Enzymology, 194, 182-187(1991), etc. Transformation of insect cells or insects can be carried out in accordance with methods as disclosed in, for example, Bio/Technology, 6, 47-55, (1988).

Transformation of animal cells can be carried out by methods as disclosed in, for example, Cell Engineering, separate vol. 8, New Cell Engineering Experiment Protocol, 263-267(1995) (Shujun Company), Virology, Vol. 52, 456 (1973), etc.

In introducing the expression vector into cells, known methods such as a calcium phosphate method (Graham, F. L. and van der Eb, A. J.: Virology, 52, 456-467(1973)), an electroporation (Neumann, E. et al., EMBO Journal, 1,841-845(1982)), etc. may be used.

The transformants or transfectants wherein the expression vector carrying the DNA coding for the protein can be obtained according to the aforementioned techniques.

Examples of methods for expressing the protein of the present invention stably using animal cells are a method for selecting the cells wherein the above-mentioned expression vector is incorporated in the chromosome by means of clone selection. Briefly, the transformant is first selected using the above-mentioned selective marker as an index for selection. Then the animal cell produced as such using the selective marker is repeatedly subjected to a clone selection to give an aminal cell strain which stably exhibits a high ability of expressing the protein of the present invention. When a dhfr gene is used as a selective marker, the resisting cells are selected by a culture with a sequential increase in the MTX concentration to amplify the DNA coding for the protein of the present invention with dhfr gene in the cells whereby an animal cell strain exhibiting far higher expression can be obtained.

The protein of the present invention or a salt thereof can be also manufactured by culturing the transformant under a condition where the DNA coding for the protein of the present invention can be expressed to express and accumulate the protein of the present invention.

Culture of the transformants (transfectants) of Escherichia or Bacillus species can be carried out preferably in a liquid culture medium. The culture medium may contains carbon sources, nitrogen sources, minerals, etc. which are necessary for growing the transformants. The carbon sources may include glucose, dextrin, soluble starch, sucrose, etc. The nitrogen sources may include organic or inorganic substances such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extracts, bean-cakes, potato extracts, etc. Examples of the minerals may include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, etc. It is further allowable to add yeast extracts, vitamines, growth-promoting factors, etc. It is suitable that the pH of culture medium is at about 5 to 8.

The culture medium for Escherichia species is, for example, preferably M9 medium which contains glucose and casamino acid (Miller, Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York, (1972). If necessary, drugs such as 3β-indolyl acrylic acid can be added to the medium to improve efficiency of the promoter. In the case of Escherichia organisms as a host, the culture is carried out usually at about 15 to 43° C. for about 3 to 24 hours. As required, aeration and stirring may be applied. In the case of Bacillus organisms as a host, the culture is carried out usually at about 30 to 40° C. for about 6 to 24 hours. As required, aeration and stirring may also be applied.

In the case of yeast transformants, the culture medium used may include, for example, Burkholder minimum medium (Bostian, K. L. et al., Proceedings of the National Academy of Sciences of the United State of America, Vol. 77, 4505 (1980)), SD medium containing 0.5% casamino acid (Bitter, G. A. et al., Proceedings of the National Academy of Sciences of the United State of America, Vol. 81, 5330 (1984)), etc. It is preferable that the pH of the culture medium is adjusted to be from about 5 to 8. The culture is carried out usually at about 20 to 35° C. for about 24 to 72 hours. As required, aeration and stirring may be applied.

In the case of the transformants (or transfectants) of insect cells or insects, the culture medium used may include the Grace's insect medium supplemented with additives such as inactivated 10% bovine serum (Grace, T. C. C., Nature, 195, 788 (1962)). It is preferable that the pH of the culture medium is adjusted to be about 6.2 to 6.4. The culture is usually carried out at about 27° C. for about 3 to 5 days. As desired, aeration and stirring may be applied.

In the case of the transformants (or transfectants) of animal cells, the culture medium used may include MEM medium (Science, Vol. 122, 501 (1952)), DMEM medium (Virology, Vol. 8, 396 (1959)), RPMI 1640 medium (Journal of the American Medical Association, Vol. 199, 519 (1967)), 199 medium (Proceedings of the Society of the Biological Medicine, Vol. 73, 1 (1950)), etc. which are containing, for example, about 5 to 20% of fetal calf serum. It is preferable that the pH is from about 6 to 8. The culture is usually carried out at about 30 to 40° C. for about 15 to 72 hours. As required, medium exchange, aeration and stirring may be applied. Especially when CHO (dhfr⁻) cells and dhfr selective marker gene are used, it is preferred to use a DMEM medium containing a dialyzed fetal bovine serum which rarely contains thymidine.

Separation and purification of the protein from the above-mentioned cultures can be carried out according to methods described herein below.

To extract the protein from the cultured microorganisms, insects or cells, the microorganisms or cells are collected by known methods after the culture, suspended in a suitable buffer solution, disrupted by ultrasonic waves, lysozyme and/or freezing and thawing, etc. and, then, a crude protein extract is obtained by centrifugation or filtration. Other conventional extraction or isolation methods can be applied. The buffer solution may contain a protein-denaturing agent such as urea or guanidine hydrochloride or a surfactant such as Triton X-100™.

In the case where proteins are secreted into culture media, supernatants are separated from the microorganisms or cells after culture and collected by known methods. The culture supernatant containing the protein can be purified by suitable combinations of known methods for separation, isolation and purification. The known methods of separation, isolation and purification may include methods which utilizes solubility, such as salting out or sedimentation with solvents, methods which utilizes chiefly a difference in the molecular size or weight, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in the electric charge, such as ion-exchange chromatography, methods utilizing specific affinity such as affinity chromatography, methods utilizing a difference in the hydrophobic property, such as reversed-phase high-performance liquid chromatography, and methods utilizing a difference in the isoelectric point such as isoelectric electrophoresis, etc.

In cases where the protein thus obtained is in a free form, the free-form protein can be converted to a salt thereof by known methods or method analogous thereto. In case, where the protein thus obtained is in a salt form vice versa, the protein salt can be converted to a free form or to any other salt thereof by known methods or method analogous thereto.

The protein produced by the transformant can be arbitrarily modified or a polypeptide can be partly removed therefrom, by a suitable protein-modifying enzyme before or after the purification. The protein-modifying enzyme may include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase, etc. The activity of the protein of the present invention thus obtained can be measured by binding assay with a labeled ligand or by enzyme immunoassays (enzyme linked immunoassays) using specific antibodies.

The antibodies against the protein of the present invention, its partial peptide or a salt of either of them are any antibodies such as polyclonal antibodies and monoclonal antibodies which can recognize the protein of the present invention, its partial peptide or a salt of either of them.

The antibodies against the protein of the present invention, its partial peptide or a salt of either of them (hereinafter referred to as the protein of the present invention) may be manufactured by methods per se known to those of skill in the art or methods similar thereto, using the protein of the present invention as antigen. For example, polyclonal antibodies can be manufactured by the method as given below.

Preparation of Monoclonal Antibody:

(a) Preparation of Monoclonal Antibody-Producing Cells

The protein of the present invention is administered to warm-blooded animals either solely or together with carriers or diluents to the site favorable for antibody production. In order to potentiate the antibody productivity upon the administration, complete Freund's adjuvants or incomplete Freund's adjuvants may be administered. The administration is usually carried out once every 2 to 6 weeks and 2 to 10 times in total. Examples of the applicable warm-blooded animals are monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens. The use of mice and rats is preferred.

In establishing cells which produce monoclonal antibodies, an animal with the detectable antibody titer is selected from animals (e.g. mice) immunized with antigens, then spleen or lymph node is collected after 2 to 5 days from the final immunization and antibody-producing cells contained therein are fused with myeloma cells derived from homogeneous or heterogeneous animals to obtain monoclonal antibody-producing hybridomas. Measurement of the antibody titer in antisera may, for example, be carried out by reacting a labeled protein, which will be mentioned later, with the antiserum followed by measuring the binding activity of the labeling agent with the antibody. The cell fusion may be carried out, for example, by a method of Koehler and Milstein (Nature, 256, 495, 1975). Examples of the fusion accelerator are polyethylene glycol (PEG), Sendai virus, etc. and the use of PEG is preferred.

Examples of the myeloma cells are those derived from warm-blooded animals such as NS-l, P3U1, SP2/0, AP-1, etc. and the use of P3U1 is preferred. The preferred fusion ratio of the numbers of antibody-producing cells used (spleen cells) to the numbers of myeloma cells is within a range of about 1:1 to 20:1. When PEG (preferably, PEG 1000 to PEG 6000) is added in a concentration of about 10 to 80% followed by incubating at 20 to 40° C., preferably, at 30 to 37° C., for 1 to 10 minutes, an efficient cell fusion can be carried out.

Various methods may be applied for screening a hybridoma which produces a monoclonal antibody. For example, a supernatant of hybridoma culture is added to a solid phase (e.g. microplate) to which the protein antigen is adsorbed either directly or with a carrier, then anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used when the cells used for the cell fusion are those of mouse) which is labeled with a radioactive substance, an enzyme or the like, or protein A is added thereto and then monoclonal antibodies bound on the solid phase are detected; or a supernatant of the hybridoma culture is added to the solid phase to which anti-immunoglobulin or protein A is adsorbed, then the protein labeled with a radioactive substance or an enzyme is added and monoclonal antibodies bound with the solid phase is detected.

Selection and cloning of the monoclonal antibody-producing hybridoma may be carried out by methods per se known to those of skill in the art or methods similar thereto. Usually, it is carried out in a medium for animal cells, containing HAT (hypoxanthine, aminopterin and thymidine). With respect to a medium for the selection, for the cloning and for the growth, any medium may be used so far as hybridoma is able to grow therein. Examples of the medium are an RPMI 1640 medium (Dainippon Pharmaceutical Co., Ltd., Japan) containing 1 to 20% (preferably 10 to 20%) of fetal calf serum (FCS), GIT medium (Wako Pure Chemical, Japan) containing 1 to 20% of fetal calf serum and a suitable serum-free medium for hybridoma (SFM-101; Nissui Seiyaku, Japan). The culture temperature is usually 20 to 40° C. and, preferably, about 37° C. The culture period is usually from five days to three weeks and, preferably, one to two weeks. The culture is usually carried out in 5% carbon dioxide gas. The antibody titer of the supernatant of the hybridoma culture may be measured by the same manner as in the above-mentioned measurement of the antibody titer in the antiserum.

(b) Purification of the Monoclonal Antibody

The separation and purification of the monoclonal antibody may be carried out by methods for separating/purifying immunoglobulin such as salting-out, precipitation with alcohol, isoelectric precipitation, electrophoresis, adsorption/deadsorption using ion exchangers such as DEAE, ultracentrifugation, gel filtration, specific purifying methods in which only an antibody is collected by treatment with an active adsorbent such as an antigen-binding solid phase, protein A or protein G and the bond is dissociated whereupon the antibody is obtained.

Preparation of a Polyclonal Antibody:

The polyclonal antibody of the present invention can be produced by per se known methods or methods analogous thereto. The method comprises preparing an immunogen (antigen protein) per se or a conjugate of an imunogen with a carrier protein, immunizing a warm-blooded animal in the same manner as described for the production of the monoclonal antibody, harvesting a fraction containing the antibody against the protein of the present invention from the immunized animal, and purifying the harvested antibody.

Referring to the immunogen-carrier protein conjugate for use in the immunization of a warm-blooded animal, the kind of carrier protein and the ratio of the carrier and hapten are not particularly restricted only if the production of the antibody against the hapten conjugated with the particular carrier protein and used for immunization proceeds efficiently. Thus, for example, bovine serum albumin, bovine thyroglobulin, hemocyanine, or the like is coupled in the weight ratio of about 0.1 to 20, preferably about 1 to about 5, to unity of the hapten.

A variety of condensing agents can be used for this coupling between the hapten and the carrier. Thus, for example, a glutaraldehyde, carbodiimide, maleimide, or a thiol or dithiopyridyl group-containing active ester reagent can be employed.

The condensation reaction product is administered to a warm-blooded animal at a site favorable for antibody production, either as it is alone or together with a carrier or diluent. Enhancing antibody production, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered. Administration is carried out generally once in about 2 to 6 weeks for a total of about 3 to 10 times.

The polyclonal antibody can be harvested from the blood, ascites fluid, or other body fluid, preferably from the blood, of the host warm-blooded animal.

The polyclonal antibody titer in the antiserum can be determined in the same manner as the determination of monoclonal antibody described hereinbefore. The separation and purification of the polyclonal antibody can be carried out by the same method as that described for the separation and purification of monoclonal antibody.

The antisense DNA having a nucleotide sequence complementary or substantially complementary to the DNA coding for the protein or the partial peptide of the present invention (hereinafter referred to as the DNA of the present invention) can be any antisense DNA having a nucleotide sequence complementary or substantially complementary to that of the DNA of the present invention and capable of suppressing expression of the DNA.

The nucleotide sequence substantially complementary to the DNA of the present invention may, for example, be a nucleotide sequence having an identity of not less than about 70%, preferably not less than about 80%, more preferably not less than about 90%, and for still better results, not less than about 95% to the total nucleotide sequence or partial nucleotide sequence of the nucleotide sequence complementary to that the DNA of the present invention. Particularly preferred is an antisense DNA having an identity of not less than about 70%, preferably not less than about 80%, and more preferably not less than about 90%, and for still better results, not less than about 95% to the nucleotide sequence of the domain, of the complete nucleotide sequence complementary to that of the DNA of the invention, which encodes the N-terminal region of the protein of the present invention (e.g. the nucleotide sequence of the domain around the initiation codon). The antisense DNA can be synthesized using a known DNA synthesis hardware.

The protein of the present invention is a metalloprotease (preferably human-liver metalloprotease) such that the molecular weight of its proteinaceous domain is about 2 to 7×10⁴ Da, preferably about 2 to 6×10⁴ Da, and the molecular weight of its proteolytic domain is about 2 to 5×10⁴ Da, the activity of which is elevated in, for example, ovulation, development and differentiation, osteogenesis, atretic uterus, and vascularization. Moreover, its activity is increased in, for example, rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), liver disease such as hepatolienal fibrosis and cirrhosis, peridontitis, pulmonary fibrosis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature delivery, and atherosclerosis. On the other, its activity is suppressed in, for example, diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, herniated disk, etc.

Uses for the protein of the present invention or its partial peptide thereof, or a salt of either of them (hereinafter sometimes referred to briefly as the protein of the present invention), the DNA coding for the protein or its partial peptide of the invention (hereinafter sometimes referred to briefly as the DNA of the present invention), the antibody against the protein of the present invention (hereinafter sometimes referred to as the antibody of the present invention), and the antisense DNA are now described.

-   (1) Therapeutic or prophylactic composition for various diseases     with which the protein of the present invention is associated

In the event of an abnormality or defect in the DNAs of metalloproteases, or when the expression or the activity of metalloproteases is suppressed, various diseases such as diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis and herniated disk are induced.

Therefore, the protein or the DNA of the present invention can be used as a pharmaceutical composition such as a therapeutic or prophylactic composition for a variety of diseases which associates with an abnormal expression or activity of metalloprotease such as diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, herniated disk, and so on.

For example, when there is a patient whose signal transductions in cells cannot function sufficiently or normally because of a decrease or a defect in the metalloproteases in vivo, the role of the metalloproteases for said patient can be expected sufficiently or normally by:

-   (a) administering the DNA coding for the protein of the present     invention to the patient to express it; -   (b) inserting the DNA coding for the protein of the present     invention into cells to express it and transplanting the cells to     said patient, or -   (c) administering the protein to the patient.

When the DNA of the present invention is used as the above-mentioned pharmaceutical composition, said DNA may be used alone or after inserting it into a suitable vector such as retrovirus vector, adenovirus vector, adenovirus-associated virus vector, pox virus etc. followed by subjecting the product vector to a conventional means. The DNA can also be administered as “naked” DNA, with physiologically acceptable carriers such as adjuvants to assist in uptake, by “gene” gun or by a catheter such as a catheter with a hydrogel.

If one wishes to use the protein of the present invention, one would use it in a purified form, preferably in a purity of at least 90%, more preferably at least 95%, still more preferably at least 98% and most preferably at least 99%.

For example, the protein of the present invention can be used orally in the form of tablets which may be sugar coated as necessary, capsules, elixirs, microcapsules etc., or non-orally in the form of injectable preparations such as aseptic solutions and suspensions in water or other pharmaceutically acceptable liquids. These preparations can be produced by mixing the protein of the present invention with physiologically acceptable carriers, flavoring agents, excipients, vehicles, antiseptics, stabilizers, binders etc. in unit dosage forms required for generally accepted manners of pharmaceutical preparation. Active ingredient contents in these preparations are set so that an appropriate dose within the specified range is obtained.

Additives which can be mixed in tablets, capsules etc. include binders such as gelation, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose and saccharin, and flavoring agents such as peppermint, akamono oil and cherry. When the unit dosage form is the capsule, the above-mentioned materials may further incorporate liquid carriers such as oils and fats. Sterile compositions for injection can be formulated by ordinary methods of pharmaceutical preparation such as by dissolving or suspending active ingredients, naturally occuring vegetable oils such as sesame oil and coconut oil, etc. in vehicles such as water for injection to create pharmaceutical compositions.

Aqueous liquids for injection include physiological saline and isotonic solutions containing glucose and other auxiliary agents, e.g., D-sorbitol, D-mannitol and sodium chloride, and may be used in combination with appropriate dissolution aids such as alcohols, e.g., ethanol, polyalcohols, e.g., propylene glycol and polyethylene glycol, nonionic surfactants, e.g., polysorbate 80™ and HCO-50 etc. Oily liquids include sesame oil and soybean oil, and may be used in combination with dissolution aids such as benzyl benzoate and benzyl alcohol. Furthermore the above-mentioned materials may also be formulated with buffers, e.g., phosphate buffer and sodium acetate buffer; soothing agents, e.g., benzalkonium chloride, procaine hydrochloride; stabilizers, e.g., human serum albumin, polyethylene glycol; preservatives, e.g., benzyl alcohol, phenol; antioxidants etc. The thus-prepared pharmaceutical composition such as an injectable liquid is normally filled in an appropriate ampule.

The vector comprising the DNA of the present invention can be formulated as well as mentioned above, and usually can be used non-orally.

Because the thus-obtained preparation is safe and of low toxicity, it can be administered to humans or mammals (e.g., rat, mouse, guinia pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).

The dose of the protein of the present invention may vary depending on subject disease, subject of administration, way of administration, and so on. When the protein of the present invention is used, for example, for treating diabetic nephropathy by oral administration, the dose of the protein of the present invention is normally about 0.1 to 100 mg, preferably 1.0 to 50 mg, and more preferably 1.0 to 20 mg per day for an adult (weighing 60 kg). When the protein of the present invention is used, for example, for treating herniated disk by non-oral administration, it is advantageous to administer the protein of the present invention to the diseased part in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding does as converted per 60 kg weight can be administered.

-   (2) Screening of compounds as candidates which are medicinally     useful against diseases

Any compounds or their salts which activate the function, for example a proteolytic activity, of the protein of the present invention can be used as a pharmaceutical composition such as a therapeutic or prophylactic composition for diseases such as diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, and herniated disk. Therefore, the protein of the present invention is useful as a screening reagent for compounds or their salts, which activate the function of the protein of the present invention.

On the other hand, any compounds or their salts which inhibit the function of the protein of the present invention can be used as a pharmaceutical composition such as a therapeutic or prophylactic composition for wound, rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), liver disease such as hepatolienal fibrosis and cirrhosis, peridontitis, pulmonary fibrosis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature delivery, atherosclerosis, septemia, multiple (disseminated) sclerosis, cachexia, hypercalcemia, leukemia, lymphoma, diabetes, autoimmune diseases such as systemic lupus erythematosus, asthma, immune diseases such as allergic rhinitis and atopic dermatitis, generalized inflammatory reactions associated with trauma, burn or acute pancreatitis, ischemia-reperfusion syndrome, cardiovascular diseases such as myocardial infarction, congestive heart failure, etc., organ transplantation and graft-vs.-host disease (GVHD). Therefore, the protein of the present invention is useful as a screening reagent for those compounds or their salts which inhibit the function of the protein of the present invention.

The present invention, therefore, provides

-   (1) a method for screening for a compound which activates the     function (e.g. a proteolytic activity) of the protein of the present     invention or its partial peptide, or a salt of either of them (such     compound will sometimes be referred to as activator), or a compound     which inhibits the function of the protein of the present invention     or its partial peptide thereof, or a salt of either of them (such     compound will sometimes be referred to as inhibitor) characterized     in that the protein of the present invention or its partial peptide,     or a salt of either of them, is used as a screening reagent.

More particularly, the invention provides

-   (2) a method for screening for the activator or inhibitor,     characterized by comparing the results in cases of (i) a substrate     is contacted with the protein of the present invention and (ii) a     substrate and a test compound are contacted with the protein of the     present invention.

More specifically, the above screening method is characterized by measuring and comparing the proteolytic activity of the protein of the present invention in cases of (i) and (ii).

The substrate may include any substances which may function as substrates for the protein of the present invention. Examples of the substrate are casein, azocasein, FITC-casein, radio(e.g. ¹⁴C, ³H, etc.) labeled casein, collagen, azocollagen, FITC-collagen, radio(¹⁴C, ³H, etc.)-labeled collagen, and oligopeptides having a (7-methoxycoumarin-4-yl)acetyl group in the N-terminal domain and an N³-(2,4-dinitrophenyl)-2,3-diaminopropionyl group at a position towards the C-terminus by a few residues from the position where the first mentioned group is attached.

Examples of the test compound that can be used includes but is not limited to peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, and animal tissue extracts. Such compounds may be novel substances or known substances.

For carrying the above screening method into practice, the protein of the present invention is first suspended in a suitable screening buffer to prepare a sample. The buffer may be any buffer that does not affect the binding of the protein of the present invention to the substrate, such as a phosphate buffer or Tris-HCl buffer in a pH range of about 4 to 10 (preferably pH about 6 to 8).

The proteolytic activity of the protein of the present invention can be determined by the known PER method (F. T. Lundy et al. Electrophoresis, 16, 43, 1995), and so on. Specifically, any test compounds that activate the proteolytic activity by not less than about 20%, preferably not less than 30%, more preferably not less than 50%, in experiment (ii) as compared with experiment (i) can be selected as an activator of the proteolytic activity of the protein of the present invention, while any test compounds that inhibit the proteolytic activity by not less than about 20%, preferably not less than 30%, more preferably not less than 50%, in experiment (ii) as compared with experiment (i) can be selected as an inhibitor of the proteolytic activity of the protein of the present invention.

The screening kit of the present invention comprises the protein of the present invention or peptide, or a salt of either of them. The following is a typical screening kit embodying the principle of the present invention.

Screening Reagents:

(1) Screening buffer

Tris-HCl buffer, pH 8.0

(sodium chloride and calcium chloride contained)

(2) Protein sample

The protein of the invention or its partial peptide

(3) Substrate

Casein 20 mg/ml

(4) Detection

Coomassie Brilliant Blue (CBB) staining Assay Protocol:

Add aminophenyl mercuric acetate (final concentration 1 mM) to the protein of the present invention and react at 37° C. for 30 minutes. Electrophorese the reaction mixture on SDS-polyacrylamide gels (non-reducing) in accordance with PER (F. T. Lundy et al., Electrophoresis, 16, 43, 1995). Then, saturate the polyacrylamide gels with the substrate and react in the reaction medium at 37° C. for 16 hours. After the reaction, stain the gels with CBB to detect a proteolytic activity.

The compound or a salt thereof which can be identified by the screening method of the present invention or by using the screening kit of the present invention is a compound selected from among a peptide, protein, nonpeptide compound, synthetic compound, fermentation product, cell extract, plant extract, or animal tissue extract, which activates or inhibits the function of the protein of the present invention.

The salt of the compound may be the same those as mentioned above as to the protein of the present invention.

The compound which activates the function (e.g. proteolytic activity) of the protein of the present invention is safe and of low toxicity, and can be used as therapeutic and prophylactic composition for various diseases such as diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, herniated disk and so on.

On the other hand, the compound which inhibits the function (e.g. proteolytic activity) of the protein of the present invention is safe and of low toxicity, and can be used as therapeutic and prophylactic composition for various diseases such as wound, rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), liver disease such as hepatolienal fibrosis and cirrhosis, peridontitis, pulmonary fibrosis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature delivery, atherosclerosis, septemia, multiple (disseminated) sclerosis, cachexia, hypercalcemia, leukemia, lymphoma, diabetes, autoimmune diseases such as systemic lupus erythematosus, asthma, immune diseases such as allergic rhinitis and atopic dermatitis, generalized inflammatory reactions associated with trauma, burn or acute pancreatitis, ischemia-reperfusion syndrome, cardio-vascular diseases such as myocardial infarction, congestive heart failure, etc., organ transplantation, graft-vs.-host disease (GVHD) and so on.

The compound which is identified by the screening method or the screening kit can be used as the above-mentioned therapeutic or prophylactic composition in accordance with a conventional means. The compound can be used in the form of tablets, capsules, elixirs, microcapsules, aseptic solutions, suspensions and so on as well as the pharmaceutical composition comprising the protein of the present invention as mentioned above.

Because the thus-obtained preparation is safe and of low toxicity, it can be administered to humans or mammals (e.g., rats, rabbits, sheep, pigs, bovines, cats, dogs, monkeys, etc.).

The dose of the compound or a salt thereof may vary depending on subject disease, subject of administration, way of administration, and so on. When the compound inhibiting the function of the protein of the present invention is used, for example, for treating diabetic nephropathy by oral administration, the dose of the compound is normally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for an adult (weighing 60 kg). When the compound inhibiting the function of the protein of the present invention is used, for example, for treating diabetic nephropathy by non-oral administration, it is advantageous to administer the compound in the form of injectable preparation at a daily dose of about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per administration by an intravenous injection for an adult (weighing 60 kg), depending on subject of administration, subject disease and so on. For other animal species, corresponding does as converted per 60 kg weight can be administered.

-   (3) Quantitative determination of the protein of the present     invention

The antibody of the present invention is capable of specifically recognizing the protein of the present invention and, accordingly, it can be used for quantitative determination of the protein of the present invention in test liquid samples and particularly for quantitative determination by sandwich immunoassays.

Thus, the present invention provides, for example, the following methods:

-   (i) a quantitative determination of the protein of the present     invention in a test liquid sample, which comprises -   (a) competitively reacting the test liquid sample and a labeled     protein of the present invention with the antibody of the present     invention, and -   (b) measuring the ratio of the labeled protein of the present     invention binding with said antibody; and -   (ii) a quantitative determination of the protein of the present     invention in a test liquid sample, which comprises -   (a) reacting the test liquid sample with an antibody immobilized on     an insoluble carrier and a labeled antibody simultaneously or     continuously, and -   (b) measuring the activity of the labeling agent on the insoluble     carrier, wherein one antibody is capable of recognizing the     N-terminal region of the protein of the present invention while     another antibody is capable of recognizing the C-terminal region of     the protein of the present invention.

When the monoclonal antibody of the present invention recognizing a protein of the present invention (hereinafter, sometimes referred to as “monoclonal antibody of the present invention”) is used, the quantity of the protein of the present invention can be measured and, moreover, the protein of the present invention can be detected by means of a tissue staining, etc. as well. For such an object, antibody molecules per se may be used, or F(ab′)₂, Fab′ or Fab fractions of the antibody molecule may also be used. There is no particular limitation for the measuring method using the antibody of the present invention and any measuring method may be used so far as it relates to a method in which the amount of antibody, antigen or antibody-antigen complex, depending on or corresponding to the amount of antigen, e.g. the amount of the protein of the present invention in the liquid sample to be measured, is detected by a chemical or a physical means and then calculated using a standard curve prepared by a standard solution containing the known amount of antigen. For exmaple, nephrometry, competitive method, immunometric method and sandwich method are suitably used and, in terms of sensitivity and specificity, the sandwich method which will be described herein later is particularly preferred.

Examples of the labeling agent used in the measuring method using the labeling substance are radioisotopes, enzymes, fluorescent substances, luminescent substances, colloids, magnetic substances, etc. Examples of the radioisotope are [¹²⁵I], [¹³¹I], [³H] and [¹⁴C]. Preferred examples of the enzyme are those which are stable and with much specific activity, such as β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase. Examples of the fluorescent substance are fluorescamine, fluorescein isothiocyanate, etc. Examples of the luminescent substance are luminol, luminol derivatives, luciferin, lucigenin, etc. Further, a biotin-avidin system may also be used for binding an antibody or antigen with a labeling agent.

In an insolubilization (immobilization) of antigens or antibodies, a physical adsorption may be used or a chemical binding which is usually used for insolubilization or immobilization of proteins or enzymes may be used as well. Examples of the carrier are insoluble polysaccharides such as agarose, dextran and cellulose; synthetic resins such as polystyrene, polyacrylamide and silicone; glass; etc.

In a sandwich method, the test liquid is made to react with an insolubilized monoclonal antibody of the present invention (the first reaction), then it is allowed to react with an another labeled monoclonal antibody of the present invention (the second reaction) and the activity of the labeling agent on the insoluble carrier is measued whereupon the amount of the protein of the present invention in the test liquid can be determined. The first reaction and the second reaction may be conducted reversely or simultaneously or they may be conducted with an interval. The type of the labeling agent and the method of insolubilization may be the same as those mentioned hereinbefore. In the immunoassay by means of a sandwich method, it is not always necessary that the antibody used for the labeled antibody and the antibody for the solid phase is one type or one species but, with an object of improving the measuring sensitivity, etc., a mixture of two or more antibodies may be used as well.

In the method of measuring the protein of the present invention by the sandwich method of the present invention, the preferred monoclonal antibodies of the present invention used for the first and the second reactions are antibodies wherein their sites binding to the protein of the present invention are different from each other. Thus, antibodies used in the first and the second reactions are those wherein, when an antibody used in the second reaction recognizes the C-terminal region of the protein of the present invention, then another antibody recognizing the site other than C-terminal regions, e.g. recognizing the N-terminal region, is preferably used in the first reaction.

The monoclonal antibody of the present invention may be used in a measuring system other than the sandwich method such as a competitive method, an immunometric method and a nephrometry. In the competitive method, an antigen in the test solution and a labeled antigen are allowed to react with an antibody in a competitive manner, then an unreacted labeled antigen (F) and a labeled antigen (B) binding with an antibody are separated (i.e. B/F separation) and the labeled amount of any of B and F is measured whereupon the amount of the antigen in the test solution is determined. With respect to a method for such a reaction, there are a liquid phase method in which a soluble antibody is used as the antibody and the B/F separation is conducted by polyethylene glycol, a second antibody to the above-mentioned antibody, etc.; and a solid phase method in which an immobilized antibody is used as the first antibody or a soluble antibody is used as the first antibody while an immobilized antibody is used as the second antibody.

In the immunometric method, an antigen in the test solution and an immobilized antigen are subjected to a competitive reaction with a certain amount of a labeled antibody followed by separating into solid and liquid phases or the antigen in the test solution and an excess amount of labeled antibody are allowed to react, then an immobilized antigen is added to bind an unreacted labeled antibody with the solid phase and separated into solid and liquid phases. After that, the labeled amount of any of the phases is measured to determine the antigen amount in the test solution.

In the nephrometry, the amount of insoluble sediment which is produced as a result of the antigen-antibody reaction in a gel or in a solution is measured. Even when the antigen amount in the test solution is small and only a small amount of the sediment is obtained, a laser nephrometry wherein scattering of laser is utilized can be suitably used.

In applying each of those immunological measuring methods (immunoassays) to the measuring method of the present invention, it is not necessary to set up any special condition, operation, etc. therefor. A measuring system (assay system) for the protein of the present invention may be constructed taking the technical consideration of the persons skilled in the art into consideration in the conventional conditions and operations for each of the methods. With details of those conventional technical means, a variety of reviews, reference books, etc. may be referred to.

They are, for example, Hiroshi Irie (ed): “Radioimmunoassay” (Kodansha, Japan, 1974); Hiroshi Irie (ed): “Radioimmunoassay; Second Series” (Kodansha, Japan, 1979); Eiji Ishikawa et al. (ed): “Enzyme Immunoassay” (Igaku Shoin, Japan, 1978); Eiji Ishikawa et al. (ed): “Enzyme Immunoassay” (Second Edition) (Igaku Shoin, Japan, 1982); Eiji Ishikawa et al. (ed): “Enzyme Immunoassay” (Third Edition) (Igaku Shoin, Japan, 1987); “Methods in Enzymology” Vol. 70 (Immunochemical Techniques (Part A)); ibid. Vo. 73 (Immunochemical Techniques (Part B)); ibid. Vo. 74 (Immunochemical Techniques (Part C)); ibid. Vo. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)); ibid. Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)); ibid. Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (Academic Press); etc.

By using the antibody of the present invention in the above manner, the protein of the present invention can be assayed with high sensitivity.

In addition, when increase in concentration of the protein of the present invention is detected by determining the concentration of the protein of the present invention by using the antibody against the protein of the present invention, it may lead, with high probability, to the diagnosis of various diseases such as wound, rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), liver disease such as hepatolienal fibrosis and cirrhosis, peridontitis, pulmonary fibrosis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature delivery, atherosclerosis, septemia, multiple (disseminated) sclerosis, cachexia, hypercalcemia, leukemia, lymphoma, diabetes, autoimmune diseases such as systemic lupus erythematosus, asthma, immune diseases such as allergic rhinitis and atopic dermatitis, generalized inflammatory reactions associated with trauma, burn or acute pancreatitis, ischemia-reperfusion syndrome, cardiovascular diseases such as myocardial infarction, congestive heart failure, etc., organ transplantation, graft-vs.-host disease (GVHD) and so on. When decrease in concentration of the protein of the present invention is detected, it may lead, with high probability, to the diagnosis of various diseases such as diabetic nephropathy, glomerulonephritis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, herniated disk and so on.

Thus, the antibody of the present invention is useful as a diagnostic agent for the above-mentioned diseases.

Furthermore, the antibody of the present invention can be used for the purpose of detecting the protein of the present invention which may be present in test samples such as body fluids or tissues. The antibody can also be used for the construction of an antibody column for purification of the protein of the present invention, detection of the protein of the present invention in the fractions in the course of purification, and analysis of the behavior of the protein of the present invention in the test cell.

-   (4) Gene diagnostic agent

By using the DNA of the present invention as a probe, for instance, an abnormality (gene abnormality) of the DNA or mRNA coding for the protein of the present invention or its partial peptide in humans or mammals (e.g. rat, mouse, guinea pig, rabbit, sheep, swine, bovine, horse, cat, dog, monkey, chimpanzee, etc.) can be detected. Therefore, the DNA of the present invention is useful as a gene diagnostic agent for the damage to the DNA or mRNA, mutation thereof, or decreased expression thereof, or increased expression or overexpression of the DNA or mRNA.

The above-mentioned gene diagnosis using the DNA of the present invention can be carried out by, for example, the Per se known Northern hybridization assay or PCR-SSCP assay [Genomics, 5, 874-879 (1989); Proceedings of the National Academy of Sciences of the United States of America, 86, 2766-2770 (1989)].

When increase in expression of the DNA is detected by Northern hybridization assay, it may lead, with high probability, to the diagnosis of wound, rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), liver disease such as hepatolienal fibrosis and cirrhosis, peridontitis, pulmonary fibrosis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature delivery, atherosclerosis, septemia, multiple (disseminated) sclerosis, cachexia, hypercalcemia, leukemia, lymphoma, diabetes, autoimmune diseases such as systemic lupus erythematosus, asthma, immune diseases such as allergic rhinitis and atopic dermatitis, generalized inflammatory reactions associated with trauma, burn or acute pancreatitis, ischemia-reperfusion syndrome, cardiovascular diseases such as myocardial infarction, congestive heart failure, etc., organ transplantation, graft-vs.-host disease (GVHD) and so on. when decrease in expression of the DNA in detected, it may lead, with high probability, to the diagnosis of diabetic nephropathy, glomerulonephritis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis, herniated disk and so on. When a mutation of the DNA is detected by the PCR-SSCP assay, it may lead, with high probability to diagnosis of wound, rheumatoid arthritis, osteoarthritis, cancer (metastasis and invasion), liver disease such as hepatolienal fibrosis and cirrhosis, peridontitis, pulmonary fibrosis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature delivery, atherosclerosis, septemia, multiple (disseminated) sclerosis, cachexia, hypercalcemia, leukemia, lymphoma, diabetes, autoimmune diseases such as systemic lupus erythematosus, asthma, immune diseases such as allergic rhinitis and atopic dermatitis, generalized inflammatory reactions associated with trauma, burn or acute pancreatitis, ischemia-reperfusion syndrome, cardiovascular diseases such as myocardial infarction, congestive heart failure, etc., organ transplantation, graft-vs.-host disease (GVHD), diabetic nephropathy, glomerulonephritis, hepatolienal fibrosis, hepatocirrhosis, osteopetrois, herniated disk and so on.

-   (5) Antisense DNA

An antisense DNA capable of making complementary conjugate with the DNA of the present invention to suppress expression of the DNA is capable of inhibiting the function of the protein or the DNA of the present invention in the body. Therefore, the antisense DNA can be used as a pharmaceutical composition such as a therapeutic and prophylactic composition for diseases such as wound, rheumatoid arthritis, osteoarthritis, cancer (metasrasis and invasion), liver disease such as hepatolienal fibrosis and cirrhosis, periontitis, pulmonary fibrosis, corneal ulcer, gastric ulcer, myocardiopathy, aneurysm, otosclerosis, epidermolysis bullosa, premature delivery, atherosclerosis, septemia, multiple (disseminated) sclerosis, cachexia, hypercalcemia, leulemia, lymphoma, diabetes, systemic lupus erythematosus, asthma, allergic rhinitis, atopic dermatitis, trauma, burn, acute pancreatitis, ischemia-reperfusion syndrome, myocardial infarction, congrestive heart failure, organ transplantation, graft-vs.-host disease (GVHD) and so on.

The antisense DNA can be used as the above-mentioned pharmaceutical composition in the same manner as the pharmaceutical composition comprising the DNA of the present invention as mentioned above.

-   (6) Preparation of non-human animals harboring a foreign DNA coding     for the protein of the present invention

Transgenic non-human animals which express the protein of the present invention can be constructed by using the DNA of the present invention. As the species of non-human animals that can be used, a variety of mammals (e.g. rat, mouse, rabbit, sheep, swine, bovine, cat, dog, monkey, etc.), etc. (hereafterin referred to as animals) can be mentioned. Particularly preferred are mouse and rabbit.

In transferring the DNA of the present invention to a host animal, it is generally advantageous to use the DNA as a gene construct prepared by ligating the DNA downstream of a promoter capable of expressing the DNA in animal cells. For the transfer of a rabbit-derived DNA of the invention, for instance, a DNA transgenic animal for high production of the protein of the present invention can be constructed by the microinjection of, for example, the fertilized rabbit egg with a gene construct prepared by ligating the DNA of the present invention as derived from an animal having high homology therewith downstream of a promoter capable of causing the expression of the DNA of the present invention in animal cells. As for such promoters, viral promoters or ubiquitous expression promoters such as and metallothionein promoters can also be used.

The transfer of the DNA into the fertilized egg cell stage is secured in all the germ and somatic cells of the host animal. The presence of the protein of the present invention in the germ cells of the DNA-transferred animal means that all the progeny of the transgenic animal invariably harbors the protein of the present invention in their germ and somatic cells The offsprings of such an animal to which the gene has been passed on have the protein of the present invention in all of their germ and somatic cells.

The transgenic animal in which the DNA of the present invention has been expressed is confirmed to retain the gene stably by copulation and then can be bred from generation to generation as the DNA-harboring animals in the usual breeding environment. Furthermore, by mating male and female animals harboring the objective DNA, it is possible to obtain homozygotes having the introduced gene in both of the homologous chromosomes, and by mating such partners, it is possible to insure that all the progeny animals will harbor this DNA.

The animal to which the DNA of the present invention has been passed on has the protein of the present invention expressed in a high degree so that it is useful as an animal for screening for compounds and salts which would activate or inhibit the proteolytic activity of the protein of the present invention.

The animal to which the DNA of the present invention has been transferred can also be used as a source of cells for tissue culture. For example, the protein of the present invention can be studied either directly by analyzing the DNA or RNA in the tissues of a mouse to which the DNA of the present invention has been transferred or analyzing a tissue containing the protein of the present invention as expressed by the gene. It is possible to culture cells from a tissue containing the protein of the present invention by the standard tissue culture technique and, using the culture, study the functions of cells derived from even those tissues which are generally difficult to culture, such as brain or peripheral tissue cells. Furthermore, by using such cells, drugs which activate the functions of various tissues may be selected. Moreover, if a high-expression cell line is provided, it will be possible to isolate and purify the protein of the present invention from the cell line.

In the specification and drawings of the present application, the abbreviations used for bases (nucleotides), amino acids and so forth are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature or those conventionally used in the art. Examples thereof are given below. Amino acids for which optical isomerism is possible are, unless otherwise specified, in the L form.

DNA Deoxyribonucleic acid cDNA Complementary deoxyribonucleic acid A Adenine T Thymine G Guanine C Cytosine RNA Ribonucleic acid mRNA Messenger ribonucleic acid dATP Deoxyadenosine triphosphate dTTP Deoxythymidine triphosphate dGTP Deoxyguanosine triphosphate dCTP Deoxycytidine triphosphate ATP Adenosine triphosphate EDTA Ethylenediaminetetracetic acid SDS Sodium dodecyl sulfate Gly Glycine Ala Alanine Val Valine Leu Leucine Ile Isoleucine Ser Serine Thr Threonine Cys Cysteine Met Methionine Glu Glutamic acid Asp Aspartic acid Lys Lysine Arg Arginine His Histidine Phe Phenylalanine Tyr Tyrosine Trp Tryptophan Pro Proline Asn Asparagine Gln Glutamine pGlu Pyroglutamic acid Me Methyl Et Ethyl Bu Butyl Ph Phenyl TC Thiazolidine-4(R)-carboxamide

Substitution groups, protecting groups and reagents used in the specification of the present application are represented by the symbols set forth below.

Tos p-toluene sulfonyl CHO Formyl Bzl Benzyl Cl₂-Bzl: 2,6-dichlorobenzyl Bom Benzyloxymethyl Z Benzyloxycarbonyl Cl-Z 2-chlorobenzyloxycarbonyl Br-Z 2-bromobenzyloxycarbonyl Boc Tert-butoxycarbonyl DNP Dinitrophenyl Trt Trityl Bum Tert-butoxymethyl Fmoc N-9-fluorenylmethyloxycarbonyl HOBt 1-hydroxybenzotriazole HOOBt 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine HONB 1-hydroxy-5-norbornene-2,3-dicarboximide DCC Dicyclohexylcarbodiimide Cha Cyclohexyl alanyl Abu Aminobutyrate Abz 2-aminobenzoyl

Each SEQ ID NO set forth in the SEQUENCE LISTING of the specification refers to the following sequence:

SEQ ID NO:1 shows an amino acid sequence of the human liver-derived metalloprotease of the present invention (FIG. 1).

SEQ ID NO:2 shows an amino acid sequence of the rat liver-derived metalloprotease of the present invention (FIG. 6).

SEQ ID NO:3 shows an amino acid sequence of the partial peptide of the human liver-derived metalloprotease of the present invention (FIG. 2), wherein 97 amino acid residues are deleted from N-terminus of the amino acid sequence represented by SEQ ID NO:1.

SEQ ID NO:4 shows an amino acid sequence of the partial peptide of the human liver-derived metalloprotease of the present invention (FIG. 3), wherein 92 amino acid residues are deleted from N-terminus of the amino acid sequence represented by SEQ ID NO:1.

SEQ ID NO:5 shows an amino acid sequence of the partial peptide of the rat liver-derived metalloprotease of the present invention, wherein 98 amino acid residues are deleted from N-terminus of the amino acid sequence represented by SEQ ID NO:2.

SEQ ID NO:6 shows an amino acid sequence of the partial peptide of the rat liver-derived metalloprotease of the present invention, wherein 93 amino acid residues are deleted from N-terminus of the amino acid sequence represented by SEQ ID NO:2.

SEQ ID NO:7 shows a nucleotide sequence of the DNA coding for the human liver-derived metalloprotease comprising an amino acid sequence represented by SEQ ID NO:1 of the present invention.

SEQ ID NO:8 shows a nucleotide sequence of the DNA coding for the rat liver-derived metalloprotease comprising an amino acid sequence represented by SEQ ID NO:2 of the present invention.

SEQ ID NO:9 shows a nucleotide sequence of the DNA coding for the partial peptide comprising an amino acid sequence represented by SEQ ID NO:3 of the human liver-derived metalloprotease of the present invention.

SEQ ID NO:10 shows a nucleotide sequence of the DNA coding for the partial peptide comprising an amino acid sequence represented by SEQ ID NO:4 of the human liver-derived metalloprotease of the present invention.

SEQ ID NO:11 shows a nucleotide sequence of the DNA coding for the partial peptide comprising an amino acid sequence represented by SEQ ID NO:5 of the human liver-derived metalloprotease of the present invention.

SEQ ID NO:12 shows a nucleotide sequence of the DNA coding for the partial peptide comprising an amino acid sequence represented by SEQ ID NO:6 of the human liver-derived metalloprotease of the present invention.

SEQ ID NO:13 shows a nucleotide sequence of the synthetic primer used for the cloning of the DNA coding for the human liver-derived protein of the present invention in Example 1.

SEQ ID NO:14 shows a nucleotide sequence of the synthetid primer used for the cloning of the DNA coding for the rat liver-derived protein of the present invention in Example 1.

SEQ ID NO:15 shows a nucleotide sequence of the synthetic primer used for the construction of Escherichia coli exapression vector in Example 4.

SEQ ID NO:16 shows a nucleotide sequence of the synthetic primer used for the construction of Escherichia coli exapression vector in Example 4.

SEQ ID NO:17 shows a nucleotide sequence of the synthetic primer used for the cloning of the DNA coding for the rat liver-derived protein of the present invention in Example 9.

SEQ ID NO:18 shows a nucleotide sequence of the synthetic primer used for the cloning of the DNA coding for the rat liver-derived protein of the present invention in Example 9.

SEQ ID NO:19 shows a nucleotide sequence of the synthetic primer used for the cloning of the DNA coding for the rat liver-derived protein of the present invention in Example 9.

The transformant strain of Escherichia coli, designated DH10B/PTB1921, which is obtained in the Example 1 mentioned hereinafter, is on deposit under the terms of the Budapest Treaty from Apr. 22, 1996, with the NIBH under the Accession Number of FERM BP-5516. It is also on deposit from Apr. 19, 1996 with the IFO under the Accession Number of IFO 15950.

The transformant strain of Escherichia coli, designated DH10B/PTB1982, which is obtained in the Example 9 mentioned hereinafter, is on deposit under the terms of the Budapest Treaty from Apr. 9, 1997, with the NIBH under the Accession Number of FERM BP-5906. It is also on deposit from Apr. 9, 1997 with the IFO under the Accession Number of IFO 16074.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid sequence of a human liver-derived metalloprotease of the present invention.

FIG. 2 shows an amino acid sequence of a partial peptide of the human liver-derived metalloprotease of the present invention, wherein 97 amino acid residues are deleted from the N-terminus of the amino acid sequence shown in FIG. 1.

FIG. 3 shows an amino acid sequence of a partial peptide of a human liver-derived metalloprotease of the present invention, wherein 92 amino acid residues are deleted from the N-terminus of the amino acid sequence shown in FIG. 1.

FIG. 4 shows a nucleotide sequence of a DNA encoding a human liver-derived metalloprotease of the present invention.

FIG. 5 shows an electrophoretogram of Western blot analysis by using a human liver-derived metalloprotease of the present invention and the antibody against the protein. The abscissa represents a number of each samples. No. 1 shows a molecular weight marker used, No. 2 shows a culture supernatant of non-infected HighFive cells, No. 3 shows a culture supernatant of HighFive cells infected with β-galactosidase-expressing recombinant virus (Invitrogen), and No. 4 shows a culture supernatant of HighFive cells infected with human liver-derived metalloprotease-expressing recombinant virus. The ordinate represents a distance of electrophoretic migration (cm).

FIG. 6 shows an amino acid sequence of a rat liver-derived metalloprotease of the present invention and a nucleotide sequence of a DNA containing a DNA coding for the amino acid sequence.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLES

The following examples are intended to illustrate the present invention in further detail and should by no means be construed as defining the scope of the invention. Incidentally, the gene manipulations using Escherichia coli were made according to the protocol described in Molecular Cloning.

Example 1

Cloning of a Gene Coding for Human Liver-Derived Metalloprotease

The cloning of the cDNA was carried out using Gene Trapper Positive Selection System (GIBCO/BRL).

Escherichia coli DH12S from a human liver-derived cDNA library (GIBCO/BRL) was cultured in Terrific Broth (12 g/l bacto-tryptone (Difco), 24 g/l bacto-yeast extract (Difco), 2.3 g/l potassium dihydrogen phosphate, 12.5 g/l potassium monohydrogen phosphate) at 30° C. for 16 hours and using Qiagen Plasmid Kit (Qiagen), a plasmid cDNA library was purified and extracted. The purified plasmid cDNA library was digested with Gene II and Exo III (both from GIBCO/BRL) to construct a single-stranded cDNA library.

On the other hand, as a probe, a synthetic oligonucleotide (SEQ ID NO:13) was used for the screening of the cDNA library. The probe was labeled by biotinylating its 3′-end with TdT and biotin-14-dCTP (GIBCO/BRL). The single-stranded cDNA library was treated at 95° C. for 1 minute and, then, quenched on ice. To this was added the biotinylated probe, and hybridization was conducted at 37° C. for 1 hour and at room temperature. After hybridization, Gene Trapper Positive Selection System magnet beads (GIBCO/BRL) were added and the mixture was allowed to stand at room temperature for 30 minutes with stirring at 2-min intervals. Thereafter, the mixture was put in Gene Trapper Positive Selection System magnet track (GIBCO/BRL) and allowed to stand for 2 minutes. The supernatant was then discarded and the magnet beads were washed with Gene Trapper Positive Selection System wash buffer. This washing with the wash buffer was repeated 3 times. The beads were then placed and allowed to sit in the magnetic track and the supernatant was discarded. Then, Gene Trapper Positive Selection System elution buffer was added and the system was allowed to stand at room temperature for 5 minutes. The system was put in the magnetic track and left standing for 5 minutes, and the supernatant DNA solution was recovered.

The synthetic oligonucleotide (SEQ ID NO:13) as the primer was put in the recovered DNA solution and the system was allowed to stand at 95° C. for 1 minute. Then, Gene Trapper Positive Selection System repair enzyme was added and the mixture was allowed to stand at 70° C. for 15 minutes to synthesize a double-stranded DNA. Using an electroporation apparatus (Bio-Rad), this synthetic double-stranded DNA was introduced into Escherichia coli DH10B.

Using the resulting transformants and, as primers, 2 oligonucleotides (SEQ ID NO:13 and NO:14), a screening by colony PCR was carried out. A colony line of amplified fragments of 510 bp formed by PCR was selected as positive clones.

The selected Escherichia coli was cultured and the DNA was extracted. The reaction was carried out using ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq DNA polymerase FS (Perkin-Elmer) and the nucleotide sequence of the cDNA fragment was determined with 377 DNA Sequencer (Perkin-Elmer). The clone obtained was found to have the 2264 bp containing the poly(A)⁺ chain and a sequence of 1524 bp as represented by SEQ ID NO:7. Encoded in this cDNA fragment was a novel metalloprotease consisting of 508 amino acid residues as represented by SEQ ID NO:1 and the active center histidine residue had also been conserved. The homology with the known human metalloproteases (e.g. MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-17, MT-MMP-1, MT-MMP-2, MT-MMP-3) at the amino acid level was as low as 30 to 36%.

The plasmid pTB1921 harboring the DNA encoding the human liver-derived metalloprotease of the invention was introduced into Escherichia coli DH10B to obtain the transformant Escherichia coli DH10B/pTB1921.

Example 2

Transient Expression of the Human Liver-Derived Metalloprotease and Preparation of a Culture Supernatant

The pTB1921 obtained in Example 1, which had been inserted into the expression plasmied pCMV·SPORT (GIBCO/BRL), was used for expression in animal cells. The COS-7 cell line (purchased from the Institute for Fermentation, Osaka) was cultured in serum-containing DMEM and subcultured on the day before introduction of the gene. When the culture became 50% confluent, the COS-7 cells were washed with serum-free DMEM, and 2.5 ml of serum-free medium was added. To this cell suspension was added TRANSFECTAM (Nippon Gene) containing 5 μg pTB1921, and the mixture was incubated under 5% CO₂ at 37° C. for 4 hours. Then, 20% bovine serum-containing DMEM (2.5 ml) was added and the mixture was further incubated for 20 hours. The medium was then replaced with serum-free DMEM and the culture supernatant was recovered 3 days later. As a control, a mock culture supernatant obtained by adding TRANSFECTAM alone in otherwise the same manner was used.

Example 3

PER Assay of the Metalloprotease Activity of the Human Liver-Derived Metalloprotease

To the cell culture supernatant obtained in Example 2 was added aminophenyl-mercuric acetate (final concentration 1 mM) and the reaction was carried out at 37° C. for 30 minutes. The activity was then determined by the PER assay (F. T. Lundy et al., Electrophoresis, 16, 43, 1995). As a result, casein hydrolyzing activity which was not found in the mock culture supernatant was detected in the culture supernatant of COS-7 cells transfected with pTB1921. It was found that this activity was inhibited by o-phenanthroline.

Example 4

Construction of the Esherichia coli Expression Vector

To generate SphI and PstI cutting sites to cDNA encoding the human liver-derived metalloprotease of the present invention, using the pTB1921 obtained in Example 1 as the template and the following two oligonucleotides

(SEQ ID NO:15) 5′-CCCGCATGCTACCTGTTGCTGGGCCGCTG-3′ (SEQ ID NO:16) 5′-AAGCTGCAGATCTACGGTCTTGCGCCTGCTACA-3′ as primers, PCR (94° C.×30 sec., 55° C.×30 sec, 72° C.×1 min., 25 cycles) was carried out in accordance with the protocol accompanying the PCR amplification kit (Takara Shuzo). After the PCR product was purified by using SpinBind PCR Purification System (FMC), it was subcloned into pCRII (Invitrogen). After confirmation of being free from error of the nucleotide sequence of metalloprotease cDNA, the cDNA was cut out with SphI and PstI and ligated to similarly treated pQE30 (Qiagen). Then, Escherichia coli JM109 (Takara Shuzo) was transfected using the ligation mixture to obtain the human liver-derived metalloprotease-expressing Escherichia coli JM109/pNHMB.

Example 5

Expression of the Recombinant Metalloprotease in Escherichia coli and its Purification

Using the Escherichia coli JM109/pNHMB as obtained in Example 4, a recombinant metalloprotease was obtained. Its expression in Escherichia coli and purification were carried out in accordance with the protocol accompanying QIAexpress System (Qiagen). As a result, the objective metalloprotease of about 46 kDa was eluted with buffer E (QIAexpress System). Then, using a dialysis membrane with a cutoff molecular weight of 12000-14000 (SPECTORAPOR), the eluate was dialyzed against buffer (0.2 M Tris-HCl (pH 9.0), 3 mM 2-mercaptoethanol, 0.3 mM 2-hydroxyethyl disulfide, 2 M urea, 0.1% Triton X-100) at 4° C. for 16 hours and, then, serially against a buffer (0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 0.1 M urea, 1 mM 2-mercaptoethanol, 0.1 mM 2-hydroxyethyl disulfide, 0.05% Triton X-100) and another buffer (0.05 M Tris-HCl (pH 8.0), 0.15 M NaCl, 0.05% Triton X-100) at 4° C. for 4 hours each. In this manner, 18.2 mg of a recombinant human liver-derived metalloprotease could be obtained from 800 ml of the Escherichia coli culture.

Example 6

Establishment of an Inhibitor Screening System

A 96-well plate (Fluoro B Plate, Dainippon Pharmaceutical) was filled in with 30 μL of buffer (0.25 M Tris-HCl (pH 8.0), 5 mM CaCl₂, 100 mM NaCl, 10 μM ZnCl₂) and 20 μL of the recombinant human liver-derived metalloprotease (2.4 mg/ml) obtained in Example 5. After 10 minutes of preincubation at 37° C., the enzymatic reaction was initiated by adding 100 μL of 10 μM substrate (DNP-Pro-Cha-Abu-Cys(Me)-His-Ala-Lys(N-Me-Abz)-NH₂; Bachem]. The reaction was conducted at 37° C. for 16 hours and, using a microplate reader (MTP-32, Corona Electronic), the intensity of fluorescence in the reaction system was measured at an exciting wavelength of 365 nm and an emission wavelength of 460 nm. Compared with the intensity of fluorescence of enzyme-free control (−20), a buffer containing the renatured recombinant metalloprotease showed a fluorescence intensity of 230. When actinonin (Peptide Institute), a metalloprotease inhibitor, was added at a varying concentration to this reaction mixture, it inhibited metalloprotease activity with a 50% inhibitory concentration of about 10 μM. It was, therefore, clear that this assay system can be used for the screening of inhibitors of the human liver-derived metalloprotease.

Example 7

Acquisition of Anti-Metalloprotease Polyclonal Antibody

The recombinant human liver-derived metalloprotease (200 μg) obtained in Example 5 was suspended in complete Freund's adjuvant and injected into a Japanese white rabbit for the first immunization dose. Then, the rabbit was boosted with a suspension of 400 μg recombinant human liver-derived metalloprotease in incomplete Freund's adjuvant 4 times at 2-week intervals. Total blood was harvested at one week after the last booster administration to obtain about 50 ml of serum.

The antibody titer was determined as follows. To a 96-well plate prepared by immobilizing 0.5 μg/well of recombinant metalloprotease and blocked with BSA, diluted rabbit serum was added and the plate was allowed to stand at room temperature for 2 hours. After washing with the PBS containing 0.1% Tween-20, anti-rabbit IgG-peroxidase (Capel) was added and the plate was allowed to stand for 2 hours. After washing, citrate-phosphate buffer containing o-phenylenediamine and hydrogen peroxide was added and a colorization reaction was carried out for 20 minutes. The reaction was stopped with 1 M sulfuric acid and using a plate reader, the colorization was read at 492 nm. As a result, an antiserum showing an antibody titer about 1×10⁴-fold higher than that of a non-immunized rabbit was obtained.

Example 8

Expression of the Human Liver-Derived Metalloprotease in Insect Cells and Western Blotting with Anti-Metalloprotease Antibody

Construction of a recombinant vaculovirus and expression in insect cells were carried out using Invitrogen's Bac-N-Blue Transfection Kit according to the accompanying protocol. The plasmid obtained by introducing metalloprotease cDNA into the SacI and PstI restriction enzyme cleavage site of the expression vector pBlueBac4 (Invitrogen) and Bac-N-Blue (Invitrogen) viral DNA were introduced into Spodoptera frugiperda (Sf-9) cells, in which recombination was allowed to take place. From the blue plaques, a recombinant virus containing the human liver-derived metalloprotease cDNA was picked and used to infect HighFive insect cells (Invitrogen). The culture supernatant of the above insect cells was Western-blotted with the anti-metalloprotease antibody obtained in Example 7. As the secondary antibody, anti-rabbit IgG-alkaline phosphatase conjugate was used, and as color reagents, 5-bromo-4-chloro-3-indolyl-1-phosphate and Nitro Blue Tetrazolium (both from Promega) were used. As shown in FIG. 5, a band of about 44 kb was observed only in the culture supernatant of the HighFive cells infected with metalloprotease-expressed recombinant virus. The above results indicated that the antibody prepared in Example 7 could recognize the human liver-derived metalloprotease of the present invention.

Example 9

Cloning of a Gene Coding for Rat Liver-Derived Metalloprotease

Synthetic oligonucleotide primer:

5′-GGCAGGGATCCAGGCTCTC-3′ (SEQ ID NO:17) 5′-TGCATCCAGGTTAGGTTC-3′ (SEQ ID NO:18) prepared based on the nucleotide sequence of the cDNA coding for the human liver-derived metalloprotease obtained in Example 1 were use for PCR with rat brain and liver cDNA libraries (GIBCO/BRL) as templates. As a result, about 400 bp fragment coding for a part of the rat liver-derived metalloprotease was amplified from both rat cDNA libraries. The DNA fragment from the brain cDNA library was subcloned into pCRII (Invitrogen) and sequenced as described in Example 1. Full-length cDNA coding for the rat liver-derived metalloprotease was obtained from the rat liver cDNA library with the synthetic oligonucleotide primer: 5′-GCCGGAGCCAGAAGATGAGG-3′ (SEQ ID NO:19) prepared based on the 400 bp fragment according to the method as described in the Example 1.

The cDNA consisted of 2,049 bp and contained 1,551 bp of open reading frame coding for 517 amino acids of rat liver-derived metalloprotease and poly(A) as shown in FIG. 6. The rat liver-derived metalloprotease is 80% identical to the human liver-derived metalloprotease at the amino acid level. Escherichia coli DH10B was transformed with the plasmid pTB1982 comprising the cDNA coding for the rat liver-derived metalloprotease to obtain the transformant: Escherichia coli DH10B/pTB1982.

Industrial Applicability

The DNA coding for the protein of the present invention can be used as a therapeutic and prophylactic composition for a variety of diseases including diabetic nephropathy, glomerulonephritis, pulmonary fibrosis, hepatolienal fibrosis, hepatocirrhosis, osteopetrosis and herniated disk. The protein of the present invention is useful as a screening reagent for any compounds which activate or inhibit the function of the protein of the present invention. In addition, the antibody against the protein of the present invention specifically recognizes the protein of the present invention and can be used in the quantitative determination of the protein of the present invention in a test fluid. 

1. An isolated DNA comprising a DNA selected from the group consisting of: (a) a DNA comprising a nucleotide sequence coding for a protein comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7; and (c) a DNA comprising a nucleotide sequence that hybridizes under stringent conditions comprising a salt concentration of 19 mM Na⁺ and a temperature of 65° C. to the nucleotide sequence set forth in SEQ ID NO: 7 and that encodes a protein having a proteolytic activity that hydrolyzes casein.
 2. The DNA according to claim 1 which contains a DNA comprising a nucleotide sequence coding for a protein comprising the amino acid sequence set forth in SEQ ID NO:1.
 3. The DNA according to claim 1, which comprises the nucleotide sequence set forth in SEQ ID NO:
 7. 4. An isolated DNA which contains a DNA comprising a nucleotide sequence coding for a metalloprotease protein having the amino acid sequence set forth in SEQ ID NO: 1, or a salt thereof.
 5. The isolated DNA according to claim 4 consisting of the nucleotide sequence set forth in SEQ ID NO:
 7. 6. A recombinant vector comprising the DNA according to claim
 1. 7. A recombinant vector comprising the isolated DNA according to claim
 4. 8. A transformed host cell comprising the recombinant vector according to claim
 6. 9. A transformed host cell carrying the recombinant vector according to claim
 7. 10. A process for producing a protein encoded by the DNA according to claim 1, or a salt thereof, which comprises culturing the transformed host cell according to claim 8 under conditions suitable for expression of said protein.
 11. A process for producing a protein encoded by the DNA according to claim 4, or a salt thereof, comprising culturing the transformed host cell of claim 9 under conditions suitable for the expression of the protein. 